In this study, we present evidence showing that leucine is involved in the upregulation of system A amino acid transport activity in the L6 rat skeletal muscle cell line. At leucine concentrations of > or = 0.05 mM, the uptake of N-methylamino-alpha-isobutyric acid (MeAIB), a paradigm system A substrate, was stimulated by up to 50%. Kinetic analysis revealed that this stimulation was a result of an increase in the maximal transport rate of MeAIB uptake, from 327 +/- 26 to 450 +/- 8 pmol.min-1.mg protein-1 after incubation of cells with leucine. No significant change in the concentration at which MeAIB transport was half maximal was observed. System A activation was biphasic, reaching an initial plateau after 3 h, with a second phase of activation being observed after 5 h. The initial activation of system A transport occurred by a mechanism distinct from that activated by insulin-like growth factor-I (IGF-I) (3 nM), since the effects of leucine and IGF-I were additive. This activation was not due to transstimulation, since 2-amino-2-norbornane-carboxylic acid, a specific system L substrate, did not stimulate system A. Leucine's keto acid, ketoisocaproic acid, prevented the activation of system A transport, whereas aminooxyacetate, a transaminase inhibitor, augmented the increase in system A activity by leucine. Both cycloheximide and actinomycin D inhibited the leucine-induced increase in MeAIB uptake. The present results indicate that leucine, or some cellular component regulated by it, is capable of stimulating system A transport through control of DNA transcription, possibly of a gene encoding either a repressor or enhancer molecule of system A or perhaps of the gene encoding system A itself.