Crawling cell motility is characteristic of most animal cells and is involved in many important biological processes such as embryogenesis, immune response, and wound healing. It involves several steps: protrusion (dynamic surface extension) at the front of the cell, attachment to the substratum, and forward translocation of the cell body accompanied by the detachment and retraction of the rear portion of the cell. Crawling motion is believed to be based on actin, a major component of the cytoskeleton, and myosin II, a motor protein that is believed to produce contraction force by moving along actin filaments. Actin and myosin II are the two major structural and force-generating components of the cell's motile machinery. Protrusion is thought to be driven by the assembly of a dense actin network that is attached to the substrate through integrin-containing adhesions. Previous studies estimated the forces generated by actin polymerization and the adhesion forces, but the net force developed by the protruding leading edge has not been yet determined. The forward translocation of the cell body is believed to depend on the interaction of myosin with the actin network, but the exact mechanisms behind this interaction are still unclear. Moreover, it is still poorly understood how the molecular reactions involved in each step of the crawling motion are coordinated to result in the integrated cellular response. In this thesis work, we have quantitatively characterized the assembly and motion of actin and myosin II in the stationary and migratory states of the cell, and during the transition between these two steady-states. We have also measured the force developed by the protruding leading edge. The experimental system used is the fish epidermal keratocytes, one of the most spectacular models of cell locomotion. These cells are characterized by fast and persistent migration, and a stable and simple cytoskeletal organization. Enhanced phase contrast, interference reflection, classical fluorescence and fluorescent speckles microscopy were used as imaging techniques along with computer-based tracking approaches. We have first studied actin movement and assembly in the lamellipodium of keratocyte (thin, sheet-like extension at the leading edge of the cell). Actin dynamics in the lamellipodia of many cell types are characterized by retrograde flow of the actin network away from the leading edge. This flow is believed to result from membrane resistance to actin assembly and contractile forces in the network, and was speculated to play a role in controlling the cell shape and motion. Retrograde flow was previously observed in every crawling cell with the exception of keratocytes. We have for the first time detected actin retrograde flow throughout the keratocyte lamellipodium at velocities of 1–3 µm/min and analyzed its organization and relation to the cell motion during both unobstructed persistent migration and events of cell collision. We have found that freely moving cells exhibited a graded flow velocity increasing toward the sides of the lamellipodium, whereas in colliding cells, the velocity decreased markedly at the site of collision, with striking alteration of the flow in other lamellipodial regions. Our findings support the universality of the flow phenomenon and indicate that maintenance of the keratocyte shape during locomotion depends on the regulation of retrograde flow and actin polymerization. We have subsequently quantitatively characterized the assembly and movement of actin and myosin II throughout the entire keratocyte. Actin exhibited a slow backward motion in the lamellipodium and a faster forward motion in the trailing cell body. The change of direction occurred along the line parallel to the leading edge of the cell just in front of the front boundary of the cell body. Myosin II also moved forward in the cell body, but no motion with respect to the substratum was detected in the lamellipodium. At the cell sides, actin and myosin II exhibited motion towards the cell center with velocities increasing with the distance from the center. Net assembly of actin occurred locally at the leading edge of the lamellipodium, and disassembly at the back of the lamellipodium. Myosin II assembled in a distributed manner in the lamellipodium and disassembled in the cell body. Eventually, a simultaneous tracking of actin and myosin II revealed that myosin II moved forward relatively to actin throughout the cell. Taken together, these results suggest a longitudinal and transverse contraction along the lamellipodium/cell body junction. To understand how the polarized patterns of the organization of actin/myosin machinery were first established, we have studied in the next part of the work random cell polarization, i.e. the spontaneous transition between the stationary and the migratory state of a cell in the absence of external directional stimuli. Cell polarization and directional locomotion are believed to involve asymmetry in actin assembly, contractility, and substrate adhesion. We have analyzed the distribution and dynamics of actin and myosin II and the dynamics of substrate adhesions during cell polarization. In the stationary state, the cells exhibited lamellipodia with a uniform rate of actin retrograde flow all around the cell perimeter. Upon initiation of directional movement, retrograde flow accelerated at the prospective rear of the cell, but the lamellipodia initially persisted all around the perimeter. The assembly of actin network also continued all around the cell. Local areas of detachment from the substrate were observed at the base of rear lamellipodia. Treatment with myosin inhibitor blebbistatin nearly completely blocked the polarization. Depolymerization of microtubules with nocodazole resulted in attenuated polarization. Taken together, these results suggest that initial polarization results from asymmetry in actin retrograde flow, which is induced by myosin-dependent contraction and stabilized by microtubules. Finally, we have estimated the force necessary to stall the protruding leading edge of the keratocytes. We arrested the leading edge of a moving cell with a hydrodynamic load generated by a fluid flow from a micropipette. The flow arrested the protrusion locally, as the cell approached the pipette, causing an arc-shaped indentation and upward folding of the leading edge. Modeling of the fluid flow gave a surprisingly low value for the arresting force of just a few piconewtons per micrometer. This small force did not abolish actin polymerization nor disrupt the adhesions formed before the arrest, but rather interfered with the weak nascent adhesions at the very front of the cell. We conclude that a weak external force is sufficient to reorient the growing actin network at the leading edge and to stall the cell protrusion. Our study of the dynamics of actin and myosin II in polarizing and migrating cells provides novel and important insights into the cell motility mechanism and establishes a fundamental quantitative data set necessary to test and improve theoretical models of cell motility.