Neutral amino acid transport was characterized in the pluripotent embryonal carcinoma (EC) cell line, OC15. Ten of the thirteen amino acids tested are transported by all three of the major neutral amino acid transport systems--A, L, and ASC--although one system may make a barely measurable contribution in some cases. The characterization of N-methyl-aminoisobutyric acid (meAIB) transport points to this model amino acid as a definitive substrate for System A transport by OC15 cells. Thus, high concentrations of meAIB can be used selectively to block System A transport, and the transport characteristics of meAIB represent system A transport. Kinetic analysis of System A, with a Km = 0.79mM and Vmax = 14.4 nmol/mg protein/5 min, suggests a single-component transport system, which is sensitive to pH changes. While proline transport in most mammalian cells is largely accomplished through System A, it is about equally divided between Systems A and ASC in OC15 cells, and System A does not contribute at all to proline transport by F9 cells, an EC cell line with limited developmental potential. Kinetic analysis of System L transport, represented by Na+-independent leucine transport, reveals a high-affinity, single-component system. This transport system is relatively insensitive to pH changes and has a Km = 0.0031 mM and Vmax = 0.213 nmol/mg protein/min. The putative System L substrate, 2-aminobicyclo-[2,2,1]heptane-2-carboxylic acid (BCH), inhibits Systems A and ASC as well as System L in OC15 cells. Therefore, BCH cannot be used as a definitive substrate for System L in OC15 cells. Phenylalanine is primarily transported by Na+-dependent Systems A and ASC (83% Na+-dependent; 73% System ASC) in OC15 cells, while it is transported primarily by the Na+-independent System L in most other cell types, including early cleavage stage mouse embryos and F9 cells. We have also found this unusually strong Na+-dependency of phenylalanine transport in mouse uterine blastocysts (82% Na+-dependent). There is no evidence for System N transport by OC15 cells, since histidine is transported primarily by a Na+-independent, BCH-inhibitable mechanism.