Neuronal phospholipase D (PLD) activity was hypothesized to be involved in vesicle trafficking and endocytosis and, possibly, transmitter release. We here report that prolonged depolarization of rat hippocampal slices by potassium chloride (KCl) or 4-aminopyridine inhibited PLD activity. Similarly, PLD activity in rat cortical synaptosomes was significantly inhibited by depolarizing agents including veratridine and ouabain. Inhibition of calcium/calmodulin kinase II (CaMKII) which positively modulates synaptosomal PLD activity [Sarri et al. (1998) FEBS Lett. 440, 287-290] by KN-62 caused a further reduction of PLD activity in depolarized synaptosomes. Depolarization-induced inhibition of PLD activity was apparently not due to transmitter release or activation of other kinases. We observed, however, that KCl-induced depolarization caused an increase of inositol phosphates and a reduction of the synaptosomal pool of phosphatidylinositol-4, 5-bisphosphate (PIP(2)). Moreover, in synaptosomes permeabilized with Staphylococcus aureus alpha-toxin, PLD activation induced by calcium was abolished by neomycin, a PIP(2) chelator. We conclude that depolarizing conditions cause an inhibition of neuronal PLD activity which is likely due to breakdown of PIP(2), a required cofactor for PLD activity. Our findings suggest that neuronal PLD activity is regulated by synaptic activity.