Phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidic acid (PA) were found to inhibit strongly the citrulline formation activity of neuronal nitric oxide synthase (nNOS; EC 220.127.116.11). Such inhibition was not observed with any other phospholipid examined. A kinetic analysis of purified nNOS showed no significant change in apparent K(m) for L-Arg or NADPH caused by these inhibitory phospholipids. Electron paramagnetic resonance analysis revealed no significant spectral perturbation of the ferriheme or flavin semiquinone upon the addition of PIP2. On the other hand, a lower enhancement of the NADPH diaphorase activity by Ca(2+)-calmodulin was observed in the presence of PIP2 and PA, and the citrulline formation activity was protected from phospholipid inhibition by preincubation with Ca(2+)-calmodulin. Moreover, trypsin digestion analysis showed that the cleavage site within the calmodulin-binding site of nNOS was specifically protected from trypsin by the addition of PIP2 and PA. These results strongly suggest that PIP2 and PA inhibit the citrulline formation activity of nNOS by blocking the interaction of the enzyme with Ca(2+)-calmodulin.