We have assessed the role of synapsins in catecholamine release by comparing the properties of exocytosis in adrenal chromaffin cells from wild-type and synapsin triple knock-out (TKO) mice. Brief depolarizations led to a greater amount of catecholamine release in chromaffin cells from TKO mice in comparison to chromaffin cells from wild-type mice. This increase in catecholamine release was due to an increased number of exocytotic events, while the properties of individual quanta of released catecholamine were unchanged. Barium ions produced similar amounts of catecholamine release from TKO and wild-type chromaffin cells, suggesting that the reserve pool of chromaffin granules is unchanged following loss of synapsins. Because expression of synapsin IIa in TKO chromaffin cells rescued the defect in depolarization-induced exocytosis, the TKO phenotype apparently results from loss of synapsin IIa. We conclude that synapsin IIa serves as a negative regulator of catecholamine release and that this protein influences exocytosis from a readily releasable pool of chromaffin granules. Further, because these defects in catecholamine release are different from those observed for glutamate and GABA release in TKO mice, we conclude that the functions of synapsins differ for vesicles containing different types of neurotransmitters.