This study was designed to investigate the sequential amino acid response to acute insulin deprivation. Male Wistar rats were made severly diabetic by the intravenous injection of streptozotocin, 150 mg/kg, and maintained on insulin for 7 days. Insulin was then withheld, and measurememts of whole blood and plasma amino acid concentrations were made at 24-h intervals until 120 h, by which time animal mortality was 80%. Alanine and the other gluogenic amino acids displayed a biphasic response to insulin deprivation, decreasing in plasma and whole blood until 72 h after the last insulin injection then increasing in concentration until 120 h. The branched-chain amino acids valine, leucine, and isoleucine remained constant until 72 h after insulin, after which time their concentrations increased dramatically. It was concluded that the first phase represented enhanced gluconeogenesis and in the second phase amino acid uptake for gluconeogenesis was exceeded by net peripheral amino acid release. Enzymatic measurements showed a progressive increase in erythrocyte;plasma distribution ratios for glutamate in ketoacidosis.