The ventricles of small mammals express mostly alpha-myosin heavy chain (alpha-MHC), a fast isoform, whereas the ventricles of large mammals, including humans, express approximately 10% alpha-MHC on a predominately beta-MHC (slow isoform) background. In failing human ventricles, the amount of alpha-MHC is dramatically reduced, leading to the hypothesis that even small amounts of alpha-MHC on a predominately beta-MHC background confer significantly higher rates of force development in healthy ventricles. To test this hypothesis, it is necessary to determine the fundamental rate constants of cross-bridge attachment (f(app)) and detachment (g(app)) for myosins composed of 100% alpha-MHC or beta-MHC, which can then be used to calculate twitch time courses for muscles expressing variable ratios of MHC isoforms. In the present study, rat skinned trabeculae expressing either 100% alpha-MHC or 100% beta-MHC were used to measure ATPase activity, isometric force, and the rate constant of force redevelopment (k(tr)) in solutions of varying Ca(2+) concentrations. The rate of ATP utilization was approximately 2.5-fold higher in preparations expressing 100% alpha-MHC compared with those expressing only beta-MHC, whereas k(tr) was 2-fold faster in the alpha-MHC myocardium. From these variables, we calculated f(app) to be approximately threefold higher for alpha-MHC than beta-MHC and g(app) to be twofold higher in alpha-MHC. Mathematical modeling of isometric twitches predicted that small increases in alpha-MHC significantly increased the rate of force development. These results suggest that low-level expression of alpha-MHC has significant effects on contraction kinetics.