Several of the adenosinetriphosphatase enzymes that are responsible for cardiac muscle contraction rely on high-energy phosphates supplied by the creatine kinase (CK) system. Experimental diabetes mellitus has been shown to cause a decrease in the maximal contractile performance of the heart. We postulated that the decrease in contractile performance may be explained in part by a decrease in CK enzyme activity. To evaluate this possibility, we determined the level of CK activity and isoenzyme distribution in ventricular homogenates from normal, diabetic, and insulin-treated diabetic rats. We found that total CK activity was decreased by 35% in diabetic hearts and that a 66% reduction in the cardiac-specific MB isoenzyme occurs. Using a cDNA probe for CK-muscle (M) RNA in Northern blot analysis, we determined that a 61.1% decrease in CK-M mRNA occurs in diabetes. Chronic insulin therapy for 1 mo restores CK-M mRNA levels and enzyme activity. In conclusion, diabetes-induced CK enzyme decreases are mediated in part by a lower level of CK-M mRNA that codes for the major CK-M subunit protein. Decreased performance of the CK system may contribute to diabetic cardiomyopathy.