In response to Ca2+ entry, several prominent brain nerve terminal phosphoproteins undergo dephosphorylation, but the relation between dephosphorylation and neurotransmitter release is unknown. Using the immunosuppressants cyclosporin A (CsA) and L-683,590 (FK-520) to inhibit specifically the Ca2+/calmodulin-dependent protein phosphatase calcineurin, we demonstrate here that Ca(2+)-dependent dephosphorylation in isolated rat brain nerve terminals (synaptosomes) is mediated by calcineurin. Pretreatment with micromolar CsA resulted in a 76-95% inhibition of stimulation-induced decreases in 32P-labeled dynamin (previously referred to as dephosphin), a phosphoprotein of M(r) = 145,000 (145-kDa protein), and a phosphoprotein of M(r) = 170,000 (170-kDa protein). Pretreatment with FK-520 also inhibited Ca(2+)-dependent dephosphorylation. Using hypotonic lysates of 32P-labeled synaptosomes, the addition of Ca2+ plus calmodulin, but not either agent alone, induced dynamin dephosphorylation. CsA and FK-520 had little to no effect on the release of glutamate induced by either K(+)-depolarization or the Ca2+ ionophore ionomycin. In contrast, calcineurin inhibition led to a substantial enhancement of glutamate release evoked by the K(+)-channel blocker 4-aminopyridine, an agent whose action most closely mimics physiological stimulation. Calcineurin inhibition had no effect on stimulation-induced changes in synaptosomal Ca2+ levels. Based on our findings, we hypothesize that Ca(2+)-dependent protein dephosphorylation resulting from calcineurin activation during physiological stimulation limits neurotransmitter release from brain nerve terminals, perhaps being dependent upon cyclic repolarization of the membrane during stimulation.