In vivo and in vitro effects of ethanol on the kinetics of insulin secretion in response to glucose and tolbutamide were studied in perifused rat islets. Phases I and II insulin response to 16.7 mM glucose was decreased 46% and 48%, respectively, in islets of rats given ethanol intragastrically 1 g/kg 1 h prior to sacrifice. Mean blood ethanol levels at the time of animal sacrifice were 19.4 mmol/l. The magnitude of insulin suppression was not significantly enhanced with higher ethanol doses, 2 or 3 g/kg, although mean blood ethanol levels increased to 25.9 and 60.3 mmol/l, respectively. Similarly, significant inhibition of both phases of insulin response to glucose occurred when ethanol 1 or 3 g/kg was given intraperitoneally instead of orally. Ethanol had no effect on insulin secretion when given orally 4 h instead of 1 h prior to islet isolation. Ethanol, 65 mmol/l, added directly to rat islets perifusate simultaneously with 16.7 mM glucose decreased both phases I and II insulin response nearly half; whereas addition of 21.7 instead of 65 mmol/l ethanol had no effect. Pre-treatment of islets with 21.7 or 65 mmol/l ethanol during 30 min basal islets perifusion period had no effect on subsequent insulin response to 16.7 mM glucose. Insulin response to 10 mM tolbutamide was decreased nearly 81% by the simultaneous presence of 65 mmol/l ethanol in islets perifusate.