Hepatocytes isolated from glucagon-treated rats contain stimulated System A activity. If these cells are placed in primary culture, the enhanced transport decays rapidly provided the culture medium contains substrate amino acids. This amino acid-dependent inactivation can be composed of trans-inhibition (protein synthesis-independent), repression (protein synthesis-dependent), or both depending on the particular substrate tested. Repression was most prominently observed with a group of small neutral amino acids that are commonly found in proteins. A strong trans-inhibition response was induced by a variety of amino acid analogs. Amino acids showing no reactivity with System A produced neither trans-inhibition nor repression. Repression of System A activity in culture was blocked by inhibitors of both RNA and protein synthesis. In contrast to inhibitors of RNA biosynthesis such as actinomycin and alpha-amanitin, inhibitors of poly(A) polymerase (cordycepin and adenine-9-beta-D-arabinopyranoside) did not prevent the inactivation of the transport activity. These results demonstrate that both the stimulation of activity and the turnover of the hepatic System A activity are controlled at the transcriptional level.