The effects of fasting, refeeding, and streptozotocin-induced experimental diabetes on free amino acid concentrations in the rat exocrine pancreas were investigated. Extracts of pancreatic tissue and plasma were analyzed using high-performance liquid chromatography (HPLC). Pancreatic and plasma concentrations of alanine were reduced in animals fasted for 24 to 72 h. Pancreatic concentrations of leucine, arginine, and glutamine were increased after fasting for 48 h, and concentrations of all essential amino acids plus the nonessential amino acids glycine, serine, taurine, and glutamine were elevated after fasting for 72 h. Refeeding 72 h fasted animals for 3 h or 24 h had a negligible effect on the plasma amino acid concentrations, but markedly lowered the concentration of essential amino acids within the pancreatic tissue. Diabetes lowered the total plasma amino acid concentration from 4.9 mM to 3.1 mM but increased the total pancreatic tissue amino acid level from 16.4 mM to 18.3 mM. Efflux of intracellular amino acids into the circulation of the isolated perfused pancreas was assessed under basal conditions and in response to a vascular amino acid challenge using HPLC. L-serine transstimulated efflux of a large number of amino acids, whereas cellular efflux was only minimally affected by L-phenylalanine. Fasting and diabetes-induced increases in essential amino acid concentrations within the pancreas may reflect decreased protein synthesis, accelerated protein catabolism, or a change in membrane transport. Altered intracellular amino acid levels may directly regulate exchange diffusion of intracellular for extracellular amino acid(s).