We determined whether a customary diet high or low in protein (1) influences postabsorptive amino acid catabolism, nitrogen (N) balance, and hepatic glucose output (HGO) in normal subjects or patients with non-insulin-dependent diabetes mellitus (NIDDM) or (2) alters blood glucose levels in NIDDM. Eight normal young adults and five obese middle-aged persons with NIDDM consumed low-protein (0.8 g/kg lean body mass [LBM]) or high-protein (3.0 g/kg LBM) diets at maintenance energy for consecutive 7-day periods. Fasting and average blood glucose and N balance were measured daily. The level of dietary protein had no effect on the basal plasma leucine rate of appearance (Ra) or urinary 3-methylhistidine excretion in either subject group. Basal leucine oxidation (and by inference, whole-body amino acid catabolism) was reduced on the low-protein diet but basal HGO was not, and although exogenous glucose effectively suppressed HGO, it did not reduce leucine oxidation with either diet. After adaptation to the low-protein diet, N balance in both the normal and NIDDM subjects was close to zero. The low-protein diet reduced the fasting and daily blood glucose of the diabetic subjects by approximately 2 mmol/L (P < .05). We conclude that physiologic variation in dietary protein does not affect basal whole-body protein turnover or HGO in either normal young adults or obese middle-aged NIDDM subjects. However, protein restriction to the level of the average daily requirement significantly reduces postabsorptive and average daily blood glucose concentrations in persons with NIDDM.