Background: The microfabricated impedance spectroscopy flow cytometer used in this study permits rapid dielectric characterization of a cell population with a simple microfluidic channel. Impedance measurements over a wide frequency range provide information on cell size, membrane capacitance, and cytoplasm conductivity as a function of frequency. The amplitude, opacity, and phase information can be used for discrimination between different cell populations without the use of cell markers. Methods: Polystyrene beads, red blood cells (RBCs), ghosts, and RBCs fixed in glutaraldehyde were passed through a microfabricated flow cytometer and measured individually by using two simultaneously applied discrete frequencies. The cells were characterized at 1,000 per minute in the frequency range of 350 kHz to 20 MHz. Results: Cell size was easily measured with submicron accuracy. Polystyrene beads and RBCs were differentiated using opacity. RBCs and ghosts were differentiated using phase information, whereas RBCs and fixed RBCs were differentiated using opacity RBCs fixed using increasing concentrations of glutaraldehyde showed increasing opacity. This increased opacity was linked to decreased cytoplasm conductivity and decreased membrane capacitance, both resulting from protein cross-linking. Conclusions: This work presents label-free differentiation of cells in an on- chip flow cytometer based on impedance spectroscopy, which will be a powerful tool for cell characterization. (c) 2005 Wiley-Liss, Inc.