To investigate the mechanism of the action of high concentration of glucose on transmitter release from neuronal cells, we examined the effect of high concentration of glucose on dopamine release from pheochromocytoma-12 (PC12) cells. When the cells were incubated with 9.0 or 13.5 mg/mL glucose (2- or 3-fold of the optimum glucose concentration for PC12 cells), dopamine release was increased in a dose-related manner. Glucose-induced increase in dopamine release was blunted by nicardipine, a Ca2+ channel blocker. Following addition of 13.5 mg/mL glucose, intracellular Ca2+ concentration was increased, which was eliminated by nicardipine. Administration of 9.0 or 13.5 mg/mL glucose induced membrane depolarization in a dose-related manner. Glucose-induced dopamine release was inhibited by pinacidil or diazoxide, adenosine triphosphate (ATP)-sensitive K+ channel (KATP channel) openers. These results suggest that a high concentration of glucose induced ATP production, which blocked the KATP channel to induce membrane depolarization, and increased intracellular Ca2+ concentration and dopamine release. When the cells were cultured with 9.0 or 13.5 mg/mL glucose for 7 days, high potassium chloride (KCl)-induced dopamine release and 45Ca2+ uptake were increased. These results suggest that long-term incubation with a high concentration of glucose increased the capacity of Ca2+ uptake to enhance depolarization-induced dopamine release from PC12 cells. These data taken together suggest that a high concentration of glucose induced activation of the Ca2+ channel to stimulate dopamine release from PC12 cells.