Cell locomotion requires rapid growth of cortical actin filaments whose barbed ends are capped in the resting cell. Phosphatidylinositol-4,5-bisphosphate (PIP2) may play a critical role as an intracellular messenger in cytoskeletal rearrangement after stimulation. We have examined the effects of PIP2 micelles on the Ca2+-independent actin filament capping activity in high speed supernatants of neutrophil lysates which we had previously demonstrated to be almost entirely due to capping protein-beta2, a homologue of cap Z. High concentrations of PIP2 totally prevented the capping of exogenous spectrin-F-actin seeds by dilute supernatants of neutrophil extracts. Capping could also be inhibited, albeit less effectively, by PIP and PI, but not by other phospholipids. When incubated with filaments in the absence of supernatant, PIP2 increased the number of growing ends. PIP2 also uncapped previously capped actin filaments, as demonstrated by incubating supernatant-capped and uncapped seeds with and without PIP2 and then comparing the initial elongation rates after addition of pyrenyl-G-actin. Incubation of capped seeds with high concentrations of PIP2 increased the number of free barbed ends to a level comparable to that of the uncapped seeds exposed to PIP2. PIP2 caused uncapping to occur too quickly to be explained simply by the off-rate of capping protein-beta2, implying that PIP2 interacted directly with capping protein on the filament ends. In fact, PIP2 transiently uncapped capped seeds in the presence of excess free capping protein. From our data, we estimate that millimolar concentrations of PIP2 (almost 100-fold higher than the amount predicted from the effective concentration in purified systems) would be required to inhibit all the capping protein-beta2 in the cytosol. This discrepancy probably results, in large part, from sequestration of PIP2 by other PIP2-binding proteins in the cytoplasm. If PIP2 mediates differential cytoskeletal growth after chemoattractant stimulation in vivo, very high concentrations may be required subjacent to the plasma membrane for regional severing and uncapping of actin filaments to occur quickly near the perturbed membrane.