The effects of exercise training on cell surface GLUT-4 in skeletal muscle of the obese (fa/fa) Zucker rat were investigated using the impermeant glucose transporter photoaffinity reagent 2-N-4-(1-azi-2,2,2-trifluoroethyl)-benzoyl-1,3-bis- (D-mannos-4-yloxy)-2-propylamine (ATB-BMPA). In the absence of insulin, 3-O-methyl-D-glucose transport activity was no different in either fast-twitch (epitrochlearis) or slow-twitch (soleus) muscles of trained and sedentary obese rats. Likewise, basal ATB-BMPA-labeled GLUT-4 was not altered in these muscles with training. In contrast, the trained group exhibited significantly greater insulin-stimulated (2 mU/ml) glucose transport activity in epitrochlearis muscles than the sedentary group (0.53 +/- 0.03 vs. 0.18 +/- 0.03 mumol.g-1 x 10 min-1 for trained and sedentary, respectively), which was paralleled by a significant enhancement of insulin-stimulated cell surface GLUT-4 (5.33 +/- 0.20 vs. 1.57 +/- 0.14 disintegrations.min-1.mg-1 for trained and sedentary, respectively). Exercise training, however, did not alter insulin-stimulated glucose transport activity or cell surface GLUT-4 in soleus muscles. Finally, exercise training did not alter the ability of muscle contraction to elevate glucose transport activity or cell surface GLUT-4 in either epitrochlearis or soleus muscles of the obese rat. These results indicate that training improves insulin-stimulated glucose transport in muscle of the obese Zucker rat by increasing GLUT-4 content and by altering the normal intracellular distribution of these transporters such that they are now capable of migrating to the cell surface in response to the insulin stimulus.