We have recently demonstrated that in rats fed a high protein, carbohydrate-free (HP) diet plasma glucagon and liver cyclic AMP are increased and as a result hepatic gluconeogenesis is stimulated. To determine whether increased protein intake or lack of dietary carbohydrate was responsible for these changes, the effects of a high fat, carbohydrate-free (HF) diet on plasma glucagon and insulin, liver cyclic AMP and hepatic gluconeogenesis were determined. Perfused livers of HF-fed rats showed greater conversion of alanine and pyruvate into carbohydrate than did control liver, although the rates were less than in HP-liver. Despite elevated gluconeogenesis in liver of HF-rats, plasma glucagon and insulin and liver cyclic AMP were normal. These findings demonstrate that some mechanism other than a rise in the level of glucagon is responsible for stimulating carbohydrate synthesis. We have postulated that acetyl CoA, which is formed in large amounts as a result of accelerated fatty acid oxidation, promotes gluconeogenesis in HF-fed rats. The observation that plasma glucagon is elevated in HP-fed but not in HF-fed rats demonstrates that protein intake and not carbohydrate restriction stimulates glucagon secretion since both diets are carbohydrate-free. Despite the lack of dietary carbohydrate, plasma glucose of HF-fed rats was normal and liver glycogen was about half that of controls. When HF animals were starved for twenty-four hours, mobilization of carbohydrate stores was diminished indicating that glucose utilization is reduced as a result of fat feeding.