Phosphoinositides are involved in a large number of processes in cells and it is very demanding to study individual protein-lipid interactions in vivo due to their rapid turnover and involvement in simultaneous events. Supported lipid bilayers (SLBs) containing controlled amounts of phosphoinositides provide a defined model system where important specific recognition events involving phosphoinositides can be systematically investigated using surface sensitive analytical techniques. The authors have demonstrated the formation and characterized the assembly kinetics of SLBs incorporating phosphatidylinositol 4,5-biphosphate (PIP2; 1, 5, and 10 wt %) and phosphoinositol-3,4,5-triphosphate (1 wt %) using the quartz crystal microbalance with dissipation monitoring and fluorescence recovery after photobleaching. An increased fraction of phosphoinositides led to a higher barrier to liposome fusion, but full fluidity for the phosphatidylcholine lipids in the formed SLB. Significantly, the majority of phosphoinositides were shown to be immobile. X-ray photoelectron spectroscopy was used for the first time to verify that the PIP2 fraction of lipids in the SLB scales linearly with the amount mixed in from stock solutions. © 2010 American Vacuum Society.