The purpose of this study was to determine whether the increase in insulin sensitivity of skeletal muscle glucose transport induced by a single bout of exercise is mediated by enhanced translocation of the GLUT-4 glucose transporter to the cell surface. The rate of 3-O-[3H]methyl-D-glucose transport stimulated by a submaximally effective concentration of insulin (30 microU/ml) was approximately twofold greater in the muscles studied 3.5 h after exercise than in those of the sedentary controls (0.89 +/- 0.10 vs. 0.43 +/- 0.05 micromol . ml-1 . 10 min-1; means +/- SE for n = 6/group). GLUT-4 translocation was assessed by using the ATB-[2-3H]BMPA exofacial photolabeling technique. Prior exercise resulted in greater cell surface GLUT-4 labeling in response to submaximal insulin treatment (5.36 +/- 0.45 dpm x 10(3)/g in exercised vs. 3.00 +/- 0.38 dpm x 10(3)/g in sedentary group; n = 10/group) that closely mirrored the increase in glucose transport activity. The signal generated by the insulin receptor, as reflected in the extent of insulin receptor substrate-1 tyrosine phosphorylation, was unchanged after the exercise. We conclude that the increase in muscle insulin sensitivity of glucose transport after exercise is due to translocation of more GLUT-4 to the cell surface and that this effect is not due to potentiation of insulin-stimulated tyrosine phosphorylation.