This study determined whether exercise training in rats would prevent the accumulation of lipids and depressed glucose utilization found in hearts from diabetic rats. Diabetes was induced by intravenous streptozotocin (60 mg/kg). Trained diabetic rats were run on a treadmill for 60 min, 27 m/min, 10% grade, 6 days/wk for 10 wk. Training of diabetic rats had no effect on glycemic control but decreased plasma lipids. In vivo myocardial long-chain acylcarnitine, acyl-CoA, and high-energy phosphate levels were similar in sedentary control, sedentary diabetic, and trained diabetic groups. The levels of myocardial triacylglycerol were similar in sedentary control and diabetic rats but decreased in trained diabetic rats. Hearts were perfused with buffer containing diabetic concentrations of glucose (22 mM) and palmitate (1.2 mM). D-[U-14C] glucose oxidation rates (14CO2 production) were depressed in hearts from sedentary diabetic rats relative to sedentary control rats. Hearts from trained diabetic rats exhibited increased glucose oxidation relative to those of sedentary diabetic rats, but this improvement was below that of the sedentary control rats. [9,10(-3)H]palmitate oxidation rates (3H2O production) were identical in all three groups. These findings suggest that exercise training resulted in a partial normalization of myocardial glucose utilization in diabetic rats.