Endogenous excretion of nitrogenous products was studied during early starvation in six healthy, nonobese subjects after six days on a well-defined diet, designed to achieve net protein balance and an adequate calorie supply. The diet contained 0.5 g myofibrillar-free protein and 35 kcal/kg body weight. The subjects then fasted for three days. Urine was collected for 24-hour periods and analyzed for urea, ammonia, 3-methylhistidine, and 1-methylhistidine. Blood glucose and serum urea levels were measured daily. In a second group of subjects, muscle biopsies for determination of free amino acid concentrations were taken in the overnight fasted state and after three days of fasting. During the period with a balanced diet, urea production fell initially and stabilized after two to three days at a level of 146±15 mmol 24 h. During the period of fasting, serum urea increased from 3.0±0.4 to a maximum value of 6.2±0.7 mmol/L and urea production rose markedly, to a peak of 293±16 mmol 24 h. Ammonia excretion was 24±2 mmol 24 h before and 71±13 mmol 24 h after three days of fasting. 3-Methylhistidine excretion was stable before fasting and then rose from 154±17 to 198±17 μmol 24 h. 1-Methylhistidine excretion was unchanged during fasting. Blood glucose levels were stable at 4.8±0.2 mmol/L before fasting and then fell to 3.7±0.3 mmol/L. Intracellular concentrations of amino acids in skeletal muscle decreased markedly during fasting; after three days of fasting the glutamine concentration had fallen by 34%. The findings indicate that hepatic gluconeogenesis accelerates during the first days of starvation and that the amino acids for gluconeogenesis during early starvation derive only in part from an increased net breakdown of skeletal muscle protein. The possibility that the remainder may originate from the free glutamine pool in muscle is considered.