Vastus lateralis muscle biopsies were obtained from endurance-trained (running approximately 50 km/wk) and untrained (no regular physical exercise) men, and the expression of an array of insulin-signaling intermediates was determined. Expression of insulin receptor and insulin receptor substrate-1 and -2 was decreased 44% (P < 0.05), 57% (P < 0.001), and 77% (P < 0.001), respectively, in trained vs. untrained muscle. The downstream signaling target, Akt kinase, was not altered in trained subjects. Components of the mitogenic signaling cascade were also assessed. Extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase expression was 190% greater (P < 0.05), whereas p38 mitogen-activated protein kinase expression was 32% lower (P < 0.05), in trained vs. untrained muscle. GLUT-4 protein expression was twofold higher (P < 0.05), and the GLUT-4 vesicle-associated protein, the insulin-regulated aminopeptidase, was increased 4.7-fold (P < 0. 05) in trained muscle. In conclusion, the expression of proteins involved in signal transduction is altered in skeletal muscle from well-trained athletes. Downregulation of early components of the insulin-signaling cascade may occur in response to increased insulin sensitivity associated with endurance training.