We investigated the effect of insulin and a hypoglycemic sulfonylurea agent glyburide on cytosolic free-Ca2+ concentrations [( Ca2+]i) in isolated rat adipocytes. Both insulin and glyburide increased [Ca2+]i in a dose-dependent manner. Half-maximal effects were seen at 0.5 ng/ml of insulin and 0.5 microM glyburide. Nifedipine (25 microM), a Ca2+-channel blocker, inhibited the effect of both agents. The effect of insulin on [Ca2+]i was 40 and 70% potentiated by ambient glucose concentrations at 180 and 300 mg/dl, respectively. Depolarizing doses of potassium (40 mM) induced an increase in cytosolic Ca2+ that was also inhibited by nifedipine. It is suggested that both insulin and glyburide increase cytosolic free Ca2+ levels at least in part by promoting Ca2+ influx through voltage-dependent Ca2+ channels.