Calf spleen profilin is shown to be an in vitro substrate of purified human placental protein kinase C (PKC), with an apparent Km of 4 microM. Phosphatidylinositol bisphosphate (PIP2) was an effective activator of the profilin phosphorylation by PKC and caused a maximum 13-fold increase of Vmax with a half maximal effect at 40 micrograms/ml. The action of PIP2 was not mimicked by phosphatidylserine, phosphatidic acid or phosphatidylinositol, whereas phosphatidylinositol monophosphate was slightly stimulatory. By contrast, protein kinase C-dependent phosphorylation of histone type III-S, myelin basic protein or lipocortin-I was not affected by PIP. It is suggested that PIP2 modifies the nature of the profilin-PKC interactions.