We tested the hypothesis that diabetes impairs myocardial glucose uptake and pyruvate oxidation under normal conditions and during a dobutamine-induced increase in work. We also tested the hypothesis that an increase in work would result in a decrease in the levels of malonyl CoA, a potent inhibitor of carnitine palmitoyltransferase I (CPT I). Streptozotocin-diabetic micropigs were compared with a nondiabetic control group (n = 8 per group). Triglyceride emulsion, glucose, and somatostatin were infused into the nondiabetic group to create an acute diabetic-like state. In accord with our hypothesis, malonyl CoA decreased significantly with dobutamine in both groups, providing a possible mechanism for increased fatty acid oxidation through relieved inhibition on CPT I. In the absence of dobutamine, glucose uptake and tracer-measured lactate uptake were decreased by 57 and 80%, respectively, in the diabetic group. Dobutamine infusion resulted in similar increases in cardiac contractility, oxygen consumption, and glucose uptake in both groups despite reductions of 50-65% in GLUT-4 and GLUT-1 protein in the diabetic group. Diabetic animals possessed a defect in myocardial pyruvate oxidation, as reflected in increased lactate production, and depressed lactate uptake and pyruvate dehydrogenase activity under control and dobutamine conditions. In conclusion, the major derangement in carbohydrate metabolism in diabetic myocardium was not in glycolysis but, rather, in pyruvate oxidation.