The changes in blood glucose and plasma free fatty acid (FFA), insulin, and human growth hormone (HGH) concentrations in response to oral and intravenous glucose and intravenous insulin were measured at 7 a.m. and 7 p.m., on both occasions after a ten hour fast, in thirteen normal males and two juvenile diabetics. Basal concentration of plasma FFA was lower and that of plasma insulin higher in the morning than in the evening. Blood glucose and plasma HGH levels did not differ at these times. In the normal subjects, glucose tolerance was diminished and plasma insulin response was delayed in the evening. The delay was significantly less with intravenous glucose than with oral glucose. When compared with the usually accepted criteria for diagnosis of diabetes mellitus, the normal subjects responded in the evening as mild diabetics. Two juvenile-onset diabetics, who had virtually no increase in plasma insulin concentrations after ingestion or injection of glucose, showed no diurnal variation in glucose tolerance. No diurnal variation was seen in the blood glucose response to intravenously injected insulin or in the plasma clearance rate of insulin; these findings suggest that impairment in glucose tolerance in the evening is due to diminished early insulin response to glucose, rather than to abnormal handling of glucose or insulin. The effect of tolbutamide, phentolamine, and theophylline on the diurnal variation of glucose tolerance was studied. In four subjects, intravenous injection of tolbutamide induced a greater insulin response and a greater fall in the blood glucose concentration in the morning than in the evening. The disappearance rate of intravenously injected glucose and both early and delayed insulin responses to the glucose were enhanced to a much greater degree in the morning than in the evening by the simultaneous intravenous injection of tolbutamide. Intravenous infusion of phentolamine, an alpha-adrenergic blocking agent, in five subjects failed to influence responses to intravenous injection of glucose. Intravenous infusion of theophylline, which inhibits cyclic 3′5′-adenosine monophosphate phosphodiesterase activity, in five subjects potentiated the plasma insulin response to intravenous glucose in the evening but not in the morning; the substance did not influence the disappearance rate of glucose.