Two mechanisms have been proposed for the modulation of skeletal muscle glucose metabolism by amino acids. Whereas studies on humans and cultured cells suggested acute insulin desensitization via mammalian target of rapamycin (mTOR) and its downstream target p70 S6 kinase (S6K), investigations using native specimens of rat muscle hinted at impairment of glucose oxidation by competition for mitochondrial oxidation. To better understand these seemingly contradictory findings, we explored the effects of high concentrations of mixed amino acids on fuel metabolism and S6K activity in freshly isolated specimens of rat skeletal muscle. In this setting, increasing concentrations of amino acids dose-dependently reduced the insulin-stimulated rates of CO(2) production from glucose and palmitate (decrease in glucose oxidation induced by addition of 5.5, 11, 22, and 44 mmol/l amino acids:--16 +/- 3, -25 +/- 7, -44 +/- 4, -62 +/- 4%; P < 0.02 each). This effect could not be attributed to insulin desensitization, because it was not accompanied by any reduction of insulin-stimulated glucose transport [+12 +/- 16, +17 +/- 22, +21 +/- 33, +13 +/- 12%; all nonsignificant (NS)] or glycogen synthesis (+1 +/- 6, -5 +/- 6, -9 +/- 8, +6 +/- 5%; all NS) and because it persisted without insulin stimulation. Abrogation of S6K activity by the mTOR blocker rapamycin failed to counteract amino acid-induced inhibition of glucose and palmitate oxidation, which therefore was obviously independent of mTOR/S6K signaling (decrease in glucose oxidation by addition of 44 mmol/l amino acids: without rapamycin, -60 +/- 4%; with rapamycin, -50 +/- 13%; NS). We conclude that amino acids can directly affect muscle glucose metabolism via two mechanisms, mTOR/S6K-mediated insulin desensitization and mitochondrial substrate competition, with the latter predominating in isolated rat muscle.