The purpose of this study was to examine the role of myosin heavy chain (MHC) in determining loaded shortening velocities and power output in cardiac myocytes. Cardiac myocytes were obtained from euthyroid rats that expressed α-MHC or from thyroidectomized rats that expressed β-MHC. Skinned myocytes were attached to a force transducer and a position motor, and isotonic shortening velocities were measured at several loads during steady-state maximal Ca²⁺ activation (P_pCa4.5). MHC expression was determined after mechanical measurements using SDS-PAGE. Both α-MHC and β-MHC myocytes generated similar maximal Ca²⁺-activated force, but α-MHC myocytes shortened faster at all loads and generated ∼170% greater peak normalized power output. Additionally, the curvature of force-velocity relationships was less, and therefore the relative load optimal for power output (F_opt) was greater in α-MHC myocytes. F_opt was 0.31 ± 0.03 P_pCa4.5 and 0.20 ± 0.06 P_pCa4.5 for α-MHC and β-MHC myocytes, respectively. These results indicate that MHC expression is a primary determinant of the shape of force-velocity relationships, velocity of loaded shortening, and overall power output-generating capacity of individual cardiac myocytes.