This study examined how the duration of experimentally induced diabetes affects myocardial metabolism. Both acutely (2-day) and chronically (30-day and 90-day) streptozocin (STZ)-diabetic rats exhibited hyperglycemia and hyperketonemia, while hyperlipemia was evident only in the chronically diabetic rats. The activity of succinate dehydrogenase was lower, whereas that of 3-hydroxyacyl-CoA-dehydrogenase was higher in the hearts of chronically diabetic rats. Although myocardial concentrations of glucose-6-phosphate, glycogen, and triacylglycerols were elevated in diabetes, the patterns of alterations differed between acute and chronic diabetes. The fructose-1,6-diphosphate/fructose-6-phosphate ratio declined progressively after STZ administration, which was not accompanied by a reciprocal increase in citrate levels, although citrate concentrations were elevated. Impaired glucose oxidation was more severe in the freshly isolated heart cells from 30-day than from 2-day diabetic rats. For a given substrate concentration, the oxidation rates of palmitate and 3-hydroxybutyrate were markedly reduced in myocytes from 30-day diabetic rats. However, they were similar to or even higher than the rates found in their control counterparts under conditions that reflected the respective in vivo concentrations of the substrates. Incubating isolated myocytes from 2-day diabetic rats in the presence of insulin only partially restored the impaired glucose oxidation. Insulin administered to the animals 4 h before the experiments restored the impaired glucose oxidation by the cells. Insulin in vitro or single injection in vivo had little or no effect on glucose oxidation in isolated myocytes from 30-day diabetic rats. Our findings suggest that the myocardium undergoes a continuous and prolonged metabolic adaption in the course of experimental diabetes.