Hearts isolated from 12-mo non-insulin-dependent diabetic rats exhibited reduced rates of contractility and relaxation. Associated with the abnormality in contractility was a redistribution in myosin isozyme content to the least active V3 form. Defects in myocardial relaxation also occurred concomitantly with impaired handling of calcium. Total tissue calcium content rose 35% in the diabetic hearts. At the same time, the activity of the pump responsible for maintaining normal cytoplasmic calcium levels was reduced. At a free calcium concentration of 2.0 microM, the rates of sarcoplasmic reticular calcium uptake and adenosinetriphosphatase activity of the diabetic hearts were decreased approximately 30%. Diastolic ventricular stiffness increased dramatically. The net result of these abnormalities in calcium metabolism is a significant impairment in mechanical performance of the diabetic heart.