Cardiac myosin binding protein C (c-MyBPC) is a thick filament protein that is expressed in cardiac sarcomeres and is known to interact with myosin and actin. While both structural and regulatory roles have been proposed for c-MyBPC, its true function is unclear; however, phosphorylation has been shown to be important. In this study, we investigate the effect of c-MyBPC and its phosphorylation on two key steps of the cross-bridge cycle using fast reaction kinetics. We show that unphosphorylated c-MyBPC complexed with myosin in 1:1 and 3:1 myosin:c-MyBPC stoichiometries regulates the binding of myosin to actin (K(D)) cooperatively (Hill coefficient, h) (K(D) = 16.44 ± 0.33 μM, and h = 9.24 ± 1.34; K(D) = 11.48 ± 0.75 μM, and h = 3.54 ± 0.67) and significantly decelerates the ATP-induced dissociation of myosin from actin (K(1)k(+2) values of 0.12 ± 0.01 and 0.22 ± 0.01 M(-1) s(-1), respectively, compared with a value of 0.42 ± 0.01 M(-1) s(-1) for myosin alone). Phosphorylation of c-MyBPC abolished the regulation of the association phase (K(1)k(+2) values of 0.32 ± 0.02 and 0.33 ± 0.01 M(-1) s(-1) at 1:1 and 3:1 myosin:c-MyBPC ratios, respectively) and also accelerated the dissociation of myosin from actin (K(1)k(+2) values of 0.23 ± 0.01 and 0.29 ± 0.01 M(-1) s(-1) at a 1:1 and 3:1 myosin:c-MyBPC ratios, respectively) relative to the dissociation of myosin from actin in the presence of unphosphorylated c-MyBPC. These results indicate a direct effect of c-MyBPC on cross-bridge kinetics that is independent of the thin filament that together with its phosphorylation provides a mechanism for fine-tuning cross-bridge behavior to match the contractile requirements of the heart.