Sex differences in cardiac function have been identified. Studies suggest that the presence of testosterone in males may contribute to the observed differences in cardiac function. Our laboratory has shown previously that testosterone treatment of gonadectomized adult male rats enhances contractility of isolated rat ventricular myocytes. In this study we tested the hypothesis that gonadectomy and hormone replacement influences contractility by altering myosin heavy chain (MHC) composition. To test this hypothesis we analyzed myosin isoform expression in ventricular myocytes isolated from castrated rats displaying a decrease in myocyte contractile velocity and compared them to castrates treated with testosterone that displayed normal myocyte shortening velocity. Sixteen weeks after castration isolated rat ventricular myocytes displayed a 90% (p < 0.001) decline in MHC-α mRNA levels and over a twofold (p < 0.01) increase in MHC-β transcripts when compared to sham-operated controls. Consistent with these changes we also observed a substantial decline in the ratio of MHC-α to MHC-β protein expression. A reversal in myosin heavy chain composition was achieved following testosterone replacement. These studies provide the first direct evidence that testosterone replacement in gonadectomized animals enhances contractility via transcriptional and translational control of myosin heavy chain composition in isolated rat ventricular myocytes. The influence of testosterone on MHC composition in males may underlie some of the observed sex differences in cardiac function.