Both neurotransmitter release and insulin secretion occur via regulated exocytosis and share a variety of similar regulatory mechanisms. It has been suggested that Src family tyrosine kinases inhibit neurotransmitter release from neuronal cells (Ohnishi et al. 2001). Thus, the potential role of Src family kinases in the regulation of insulin secretion was investigated in this study. Two structurally different inhibitors of Src family kinases, SU6656 and PP2, but not the inactive compound, PP3 enhanced Ca²⁺-induced insulin secretion in both rat pancreatic islets and INS-1 cells in a concentration-dependent and time-dependent manner. Furthermore, Src family kinase-mediated insulin secretion appears to be dependent on elevated intracellular calcium and independent of glucose metabolism, the K_ATP channel, adenylyl cyclase, classical PKC isoforms (cPKC), ERK1/2 and insulin synthesis. The sites of action for Src family kinases seem to be distal to the elevation of intracellular calcium level. These results indicate that one or more Src family tyrosine kinases exert a tonic inhibitory role on Ca²⁺-dependent insulin secretion.