We determined the interaction of exercise and diet on glucose transporter (GLUT-4) protein and mRNA expression in type I (soleus) and type II [extensor digitorum longus (EDL)] skeletal muscle. Forty-eight Sprague Dawley rats were randomly assigned to one of two dietary conditions: high-fat (FAT, n=24) or high-carbohydrate (CHO, n=24). Animals in each dietary condition were allocated to one of two groups: control (NT, n=8) or a group that performed 8 weeks of treadmill running (4 sessions week-1 of 1000 m @ 28 m min-1, RUN, n=16). Eight trained rats were killed after their final exercise bout for determination of GLUT-4 protein and mRNA expression: the remainder were killed 48 h after their last session for measurement of muscle glycogen and triacylglycerol concentration. GLUT-4 protein expression in NT rats was similar in both muscles after 8 weeks of either diet. However, there was a main effect of training such that GLUT-4 protein was increased in the soleus of rats fed with either diet (P < 0.05) and in the EDL in animals fed with CHO (P < 0.05). There was a significant diet-training interaction on GLUT-4 mRNA, such that expression was increased in both the soleus (100% upward arrowP < 0.05) and EDL (142% upward arrowP < 0.01) in CHO-fed animals. Trained rats fed with FAT decreased mRNA expression in the EDL ( downward arrow 45%, P < 0.05) but not the soleus ( downward arrow 14%, NS). We conclude that exercise training in CHO-fed rats increased both GLUT-4 protein and mRNA expression in type I and type II skeletal muscle. Despite lower GLUT-4 mRNA in muscles from fat-fed animals, exercise-induced increases in GLUT-4 protein were largely preserved, suggesting that control of GLUT-4 protein and gene expression are modified independently by exercise and diet.