Substrate regulation of System A-mediated amino acid transport was investigated in primary cultures of rat hepatocytes. Studies on the substrate specificity of trans-inhibition and repression revealed considerable differences between the two processes. Those data along with a difference in temperature sensitivity suggest that the two phenomena are not related or inter-dependent in any direct way. However, kinetic analysis indicates that both trans-inhibition and repression decrease the number of functional carriers within the plasma membrane. Cis-inhibition tests show that the hepatic System A carrier exhibits a wide degree of tolerance with regard to modification of the alpha-amino and alpha-carboxyl groups. In general, the amino acids that cause the greatest degree of trans-inhibition are only moderate cis-inhibitors of System A-mediated transport (40-60% inhibition of Na+-dependent 2-aminoisobutyric acid uptake). The substrate specificity of amino acid-induced repression appears to be similar to that for System A-mediated transport, although an amino acid can exhibit cis-inhibition of System A activity without causing repression (or trans-inhibition). For example, S-methyl-L-cysteine serves as a competitive inhibitor of System A, yet it does not cause trans-inhibition or repression to a considerable degree. However, through its cis-inhibition of transport, S-methyl-L-cysteine blocks asparagine-dependent repression, apparently by suppressing the cytoplasmic accumulation of asparagine.