It has been reported that cAMP regulates Ca(2+)-dependent exocytosis via protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac) in neurons and secretory cells. It has, however, never been clarified how regulation of Ca(2+)-dependent exocytosis by cAMP differs depending on the involvement of PKA and Epac, and depending on two types of secretory vesicles, large dense-core vesicles (LVs) and small vesicles (SVs). In this study, we have directly visualized Ca(2+)-dependent exocytosis of both LVs and SVs with two-photon imaging in mouse pancreatic beta-cells. We found that marked exocytosis of SVs occurred with a time constant of 0.3 s, more than three times as fast as LV exocytosis, on stimulation by photolysis of a caged-Ca(2+) compound. The diameter of SVs was identified as approximately 80 nm with two-photon imaging, which was confirmed by electron-microscopic investigation with photoconversion of diaminobenzidine. Calcium-dependent exocytosis of SVs was potentiated by the cAMP-elevating agent forskolin, and the potentiating effect was unaffected by antagonists of PKA and was mimicked by the Epac-selective agonist 8-(4-chlorophenylthio)-2'-O-methyl cAMP, unlike that on LVs. Moreover, high-glucose stimulation induced massive exocytosis of SVs in addition to LVs, and photolysis of caged cAMP during glucose stimulation caused potentiation of exocytosis with little delay for SVs but with a latency of 5 s for LVs. Thus, Epac and PKA selectively regulate exocytosis of SVs and LVs, respectively, in beta-cells, and Epac can regulate exocytosis more rapidly than PKA.