The inhibitors okadaic acid (OA), fostriecin (FOS) and cyclosporin A (CsA), were used to investigate the roles of protein phosphatases in regulating exocytosis in rat brain synaptosomes by measuring glutamate release and the release of the styryl dye FM 2-10. Depolarization was induced by 30 mM KCl, or 0.3 mM or 1 mM 4-aminopyridine (4AP). OA and FOS produced a similar partial inhibition of KCl- and 0.3 mM 4AP- evoked exocytosis in both assays, but had little effect upon exocytosis evoked by 1 mM 4AP. In contrast, CsA had no effect upon KCl- and 0.3 mM 4AP-evoked exocytosis, but significantly enhanced glutamate release but not FM 2-10 dye release evoked by 1 mM 4AP. None of the phosphatase inhibitors changed calcium signals from FURA-2-loaded synaptosomes either before or after depolarization. Pretreatment with 100 nM phorbol 12-myristate 13-acetate abolished the inhibitory effect of OA on exocytosis induced by 0.3 mM 4AP. Taken together, these results show that exocytosis from synaptosomes has a phosphatase-sensitive and phosphatase-insensitive component, and that there are two modes of phosphatase-sensitive exocytosis that can be elicited by different depolarization conditions. Moreover, these two modes are differentially sensitive to phosphatase 2A and 2B.