The regulation of the Ca(2+)- and phorbol ester-insensitive zeta isozyme of protein kinase C (PKC zeta) by phospholipids was studied. Phosphatidylserine (PS) stimulated the activity to the same extent as proteolysis by calpain. However, the PS stimulation was abolished by phosphatidylethanolamine (PE) or phosphatidylcholine. Phosphatidylinositol-3,4,5-P3 (PIP3) produced a large stimulation of PKC zeta in the absence or presence of PS plus PE that was equal to that seen with PS alone. In the presence of PS plus PE, PIP3 was half-maximally effective at 50 nM. Phosphatidylinositol-3,4-P2 also fully activated PKC zeta, but higher concentrations (0.5 microM) of phosphatidylinositol-3-P, phosphatidylinositol-4-P, and phosphatidylinositol-4,5-P2 produced only partial (11-30%) activation of the enzyme. In contrast, when tested with "conventional" PKC purified from rat brain, none of the inositol phospholipids produced more than one-third of the stimulation seen with PS plus Ca2+ plus phorbol ester, and there was little difference between the efficacy of PIP3 and that of the other phospholipids. PIP3 produced a marked stimulation of the autophosphorylation of PKC zeta, indicating that it interacted with the enzyme directly. These results suggest that PKC zeta may be a target for PIP3 and thus may be involved in the signaling mechanism(s) for growth factors and oncogenes that increase phosphatidylinositol 3-kinase activity.