This investigation was designed to test the hypothesis that protein feeding stimulated glucagon secretion because amino acids liberated during protein digestion function as glucagon secretagogues. Rats were fed high-protein (HP) or control diets for 9--10 days and blood taken from the aorta or portal vein (PV) at 0800, 1300, 1700, 1900, 2100, and 2300 for determination of amino acids, glucose, insulin, and glucagon. Glucose, insulin, and glucagon of control rats showed little change. In HP rats, PV glucose rose during fasting (0800-1700) and declined during feeding (1700-0800), changes that reflected alterations of glucagon and insulin secretion. PV glucagon in HP rats that was elevated 2--4 times rose during fasting, whereas PV and arterial amino acids declined. HP feeding caused enhanced glucagon release that was associated with increased amino acids in PV and arterial plasma, especially the branched-chain group. Although these findings suggest that protein feeding promotes glucagon release because branched-chain amino acids are elevated, these amino acids are known to have little effect on alpha cell function. Thus, we conclude that protein feeding influences glucagon secretion through some mechanism other than increased blood amino acid levels.