We investigated the relationship between papillary muscle function and the myosin isoenzyme pattern, collagen content, and the type of myocardial collagen in diabetic rats to elucidate the mechanism of short-term myocardial dysfunction in diabetes. Diabetes was induced in 9-week-old male Wistar rats with a single intravenous injection of streptozotocin. One-half of the diabetic rats were treated with insulin. Age-matched control rats were also studied. The time to peak tension (TPT) of isometric papillary muscle contraction, time to 1/2 relaxation, and time from the peak tension to the peak decrease in tension (TPN) were significantly prolonged in diabetic rats at 4, 8, and 12 weeks. The peak increase and decrease in tension were slower in the diabetic rats compared with control rats. The level of the myocardial myosin isoenzyme V3 was greater in diabetic rats than in control rats at each interval. Findings in insulin-treated rats were similar to those in controls. The collagen content and the ratio of type I collagen to type III collagen were similar in all groups. The V3 level was significantly correlated with mechanical parameters (TPT versus %V3: r = 0.81, p < 0.01; TPN versus % V3: r = 0.78, p < 0.01). Our findings suggest that short-term myocardial dysfunction in diabetic rats is related to changes in the myosin isoenzyme pattern.