The effects of a high protein diet on blood glucose, insulin, and glucagon secretion was studied in the peripheral blood and isolated perfused pancreas of normal rats. A balanced diet (protein 27%, carbohydrate 61%, fat 12%) and a high protein diet (protein 55%, carbohydrate 30%, fat 15%) were given ad libitum to normal rats for 2 weeks. No significant difference in calorie intake or body weight changes were observed between the two groups. Fasting blood glucose and plasma insulin concentrations in rats fed the high protein diet were significantly higher than those in rats fed the balanced diet. The fasting plasma glucagon concentration, however, was the same in both groups. In the isolated perfused pancreas, the arginine-induced insulin secretion in rats fed the high protein diet was significantly higher than that in rats fed the balanced diet, whereas the arginine-induced glucagon secretion was not different in either group. The possible mechanism by which a high protein diet causes hypersecretion of insulin in vivo and in vitro are discussed.