Basolateral amino acid transport systems have been characterized in the perfused exocrine pancreas using a high-resolution paired-tracer dilution technique. Significant epithelial uptakes were measured for l-alanine, l-serine, α-methylaminoisobutyric acid, glycine, methionine, leucine, phenylalanine, tyrosine and l-arginine, whereas l-tryptophan and l-aspartate had low uptakes. α-Methylaminoisobutyric acid transport was highly sodium dependent (81 ± 3%), while uptake of l-serine, l-leucine and l-phenylalanine was relatively insensitive to perfusion with a sodium-free solution. Cross-inhibition experiments of l-alanine and l-phenylalanine transport by twelve unlabelled amino acids indicated overlapping specificities. Unidirectional l-phenylalanine transport was saturable ( K t = 16 ± 1 mM, V max = 12.3 ± 0.4 μmol/min per g), and weighted non-linear regression analysis indicated that influx was best described by a single Michaelis-Menten equation. The V max/ K t ratio (0.75) for l-phenylalanine remainded unchanged in the presence of 10 mM l-serine. Although extremely difficult to fit, l-serine transport appeared to be mediated by two saturable carriers ( K t1 = 5.2 mM, V max1 = 7.56 μmol/min per g; K t2 = 32.8 mM, V max2 = 22.9 μmol/min per g). In the presence of 10 mM l-phenylalanine the V max/ K t ratio for the two l-serine carriers was reduced, respectively, by 79% and 50%. Efflux of transported l-[ 3H]phenylalanine or l-[ 3H]serine was accelerated by increasing perfusate concentrations of, respectively, l-phenylalanine and l-serine, and trans-stimulated by other amino acids. In the pancreas neutral amino acid transport appears to be mediated by Na +-dependent Systems A and ASC, the classical Na +-independent System L and another Na +-independent System asc recently identified in erythrocytes. The interactions in amino acid influx and efflux may provide one of the mechanisms by which the supply of extracellular amino acids for pancreatic protein synthesis is regulated.