Decreased contractility has been reported in the diabetic heart. Because a close correlation exists between contractility and the activity of Ca2+ AT Pa se of purified actinomyosin and myosin, Ca2+ ATPase activity was determined in control and diabetic rats. In control rats, actinomyosin ATPase was 0.59 ± 0.05 μmol Pi/mg protein/min and had decreased by 35% to 0.38 ± 0.04 in diabetic rats (P < 0.025). Myosin ATPase activity was 1.25 ± 0.09 μmol Pi/mg protein/min in control rats and had decreased by 45% to 0.67 ± 0.05 in diabetic animals (P < 0.01). To investigate the decrease in myosin ATPase activity further, ventricular myosin was separated by pyrophosphate polyacrylamide electrophoresis into its three authentic components, V1, V2, and V3 myosin. V1 has the highest mobility and Ca2+ ATPase activity (1.27 ± 0.3 arbitrary units) and represents 72% of control myosin, whereas V3 has the lowest mobility and Ca2+ ATPase activity (0.16 ± 0.08 units) and constitutes 13% of myosin. A marked change in the predominance of V1 and V3 myosin components occurs in diabetic rats where V3 myosin predominates, representing 68% of total myosin with V1 myosin constituting only 15%. Ca2+ ATPase activities of V1, V2, and V3 myosin in control and diabetic hearts are similar; however, the predominance of V3 myosin in diabetic rats can account for the decreased Ca2+ ATPase activity of diabetic myosin. The diabetes-induced changes in myosin ATPase activity and myosin isoenzyme distribution can be reverted to control levels by insulin administration.