Nutrient-stimulated insulin secretion is dependent upon the generation of metabolic coupling factors in the mitochondria of the pancreatic B cell. To investigate the role of Ca2+ in mitochondrial function, insulin secretion from INS-1 cells stably expressing the Ca2+-sensitive photoprotein aequorin in the appropriate compartments was correlated with changes in cytosolic calcium ([Ca2+]c) and mitochondrial calcium ([Ca2+]m). Glucose and KCl, which depolarize the cell membrane, as well as the Ca2+-mobilizing agonist, carbachol (CCh), cause substantial increases in [Ca2+]m which are associated with smaller rises in [Ca2+]c. The L-type Ca2+-channel blocker, SR7037, abolished the effects of glucose and KCl while attenuating the CCh response. Glucose-induced increases in [Ca2+]m, [Ca2+]c, and insulin secretion all demonstrate a pronounced initial peak followed by a sustained plateau. All three parameters are increased synergistically when glucose and CCh are combined. Finally, [Ca2+]m, [Ca2+]c, and insulin secretion also display desensitization phenomena following repeated additions of the three stimuli. The high sensitivity of [Ca2+]m to Ca2+ influx and the desensitization-resensitization effects can be explained by a model in which the mitochondria of INS-1 cells are strategically located to sense Ca2+ influx through plasma membrane Ca2+ channels. In conclusion, the correlation of [Ca2+]m and [Ca2+]c with insulin secretion may indicate a fundamental role for Ca2+ in the adaptation of oxidative metabolism to the generation of metabolic coupling factors and the energy requirements of exocytosis.