Aim: To characterize Ca2+-stimulated exocytosis in isolated mouse pancreatic β cells. Methods: An improved method was described for isolation of mouse pancreatic β cells by collagenase P. The Ca2+ channel current and the membrane capacitance were examined by using the whole-cell patch clamp recording technique. Results: Using depolarization and flash photolysis of caged Ca2+ to induce Ca2+-dependent exocytosis in β cell from KM mouse, we have explored the characteristics of the Ca2+ channel current and the relationship between Ca2+ signals and exocytosis. The averaged peak Ca2+ current measured at +20 mV was -60±6 pA (n=13). Conclusion: We characterized three kinetically different pools of vesicles in mouse pancreatic β cells, namely an immediately releasable pool, a readily releasable pool, and a reserve pool.