Although alanine (A) is the major substrate for hepatic gluconeogenesis, it is utilized less effectively than pyruvate or lactate, thus suggesting that glutamic alanine transaminase (GAT) regulates A conversion to glucose. To test this hypothesis incorporation of A and pyruvate into glucose, glycogen and protein by isolated livers of rats fed a high protein (HP) diet was determined and compared with that by liver of normally fed animals. Effect of HP feeding on transport of α-aminoisobutyric acid (AIB), a nonutiliz-able amino acid, into liver was investigated. Liver GAT and phosphoenolpyruvate carboxykinase (PEPCK) activities were determined. HP feeding produced a twofold increase in conversion of A to glucose when perfusate A-concentration was physiological (0.5 mM) and when excess A was present. Liver glycogen synthesis from A was less than that of glucose and was unaffected by HP feeding. Incorporation of A into protein by control and HP livers was negligible. Hepatic conversion of pyruvate to glucose was markedly enhanced by the HP diet whereas glycogen synthesis from pyruvate was limited and not affected by HP feeding. AIB transport by liver of HP fed rats was double that of control liver. Liver GAT and PEPCK activities were greatly increased by HP feeding. Increased gluconeogenesis in HP liver at physiological and excess perfusate A concentrations indicates that amino acid transport and transamination are stimulated since other investigations have demonstrated that at low A levels transport is rate limiting and at high A concentrations, transamination controls gluconeogenesis. Furthermore, we observed increased AIB transport and elevated GAT activity in HP liver, thus it is clear that the GAT does not alone control conversion of A to glucose. Increased gluconeogenesis from pyruvate and elevated PEPCK activity were found suggesting that HP feeding stimulates conversion of pyruvate to phosphoenolpyruvate. The effects produced by the HP diet also occur after glucagon administration thus HP feeding probably influences gluconeogenesis by stimulating glucagon secretion.