Estrogen has been shown to help maintain the elevated expression of the high ATPase myosin isoform, V1, present in the hearts of young rats (< 70 days of age). Because hearts of this age are still undergoing significant maturation, the current study sought to determine if estrogen similarly regulates myosin isoenzyme expression in the mature adult heart. To make this determination, ten month old retired female Sprague-Dawley rats were made estrogen-deficient by ovariectomy (OVAR, n = 8). Sham-operated (CONTR, n = 8) animals served as controls. Nine weeks later, the animals were sacrificed and left ventricular tissue collected. Crude myofibrills were isolated from these samples and electrophoretically separated into the three isoenzymatic forms of cardiac myosin (V1, V2, and V3). OVAR animals were larger than the CONTR group (p < 0.05), but heart weight/body weight ratios were not different between groups. Distribution of myosin among its three isoenzymes was similar between groups (CONTR: V1, 80%, V2, 14%; V3, 6%; OVAR: V1, 77%, V2, 16%, V3, 7%). These data demonstrate that myosin isoenzyme distribution in the adult heart is unaltered by ovariectomy, suggesting that estrogen loses its ability to regulate expression of this protein in the mature heart.