The exocytosis of insulin from pancreatic β-cells is closely related to intracellular elevation of Ca²⁺. The effects of Ca²⁺ may be mediated by Ca²⁺/calmodulin-dependent protein kinase II (CaMKII). Four subunits of CaMKII, termed α, β, γ, and δ, are encoded by distinct genes, and various isoforms of these subunits exist as different splicing variants. In the brain, phosphorylation of synapsin I by the α isoform induces neurotransmitter release. In order to clarify whether phosphorylation of synapsin I by CaMKII was involved in insulin exocytosis, we cloned the isoforms of CaMKII and synapsin I from mouse insulinoma MIN6 cells. We found that β'e and δ2 are the major isoforms of CaMKII and that synapsin Ib is a major isoform of synapsin I in MIN6 cells. It was interesting that δ2 and synapsin Ib were co-localized with insulin secretory granules in the cells. Treatment of MIN6 cells with glucose and tolbutamide rapidly activated CaMKII. Immunoblot analysis with two antibodies against synapsin I phosphorylated by CaMKII demonstrated the increase in phosphorylation of synapsin I by the secretagogues. Furthermore, the secretagogue-induced phosphorylation of synapsin I and insulin secretion were potentiated by transient overexpression of the β'e or δ2 isoform. These results suggest that activation of CaMKII and the concomitant phosphorylation of synapsin I induce insulin exocytosis from pancreatic β-cells.