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Cardiac myosin binding protein-C (cMyBP-C) is a thick filament protein that influences sarcomere stiffness and modulates cardiac contraction-relaxation through its phosphorylation. Phosphorylation of cMyBP-C and ablation of cMyBP-C have been shown to increase the rate of MgADP release in the acto-myosin cross-bridge cycle in the intact sarcomere. The influence of cMyBP-C on Pi-dependent myosin kinetics has not yet been examined. We investigated the effect of cMyBP-C, and its phosphorylation, on myosin kinetics in demembranated papillary muscle strips bearing the β-cardiac myosin isoform from nontransgenic and homozygous transgenic mice lacking cMyBP-C. We used quick stretch and stochastic length-perturbation analysis to characterize rates of myosin detachment and force development over 0–12 mM Pi and at maximal (pCa 4.8) and near-half maximal (pCa 5.75) Ca 2+ activation. Protein kinase A (PKA) treatment was applied to half the strips to probe the effect of cMyBP-C phosphorylation on Pi sensitivity of myosin kinetics. Increasing Pi increased myosin cross-bridge detachment rate similarly for muscles with and without cMyBP-C, although these rates were higher in muscle without cMyBP-C. Treating myocardial strips with PKA accelerated detachment rate when cMyBP-C was present over all Pi, but not when cMyBP-C was absent. The rate of force development increased with Pi in all muscles. However, Pi sensitivity of the rate force development was reduced when cMyBP-C was present versus absent, suggesting that cMyBP-C inhibits Pi-dependent reversal of the power stroke or stabilizes cross-bridge attachment to enhance the probability of completing the power stroke. These results support a functional role for cMyBP-C in slowing myosin detachment rate, possibly through a direct interaction with myosin or by altering strain-dependent myosin detachment via cMyBP-C-dependent stiffness of the thick filament and myofilament lattice. PKA treatment reduces the role for cMyBP-C to slow myosin detachment and thus effectively accelerates β-myosin detachment in the intact myofilament lattice. NEW & NOTEWORTHY Length perturbation analysis was used to demonstrate that β-cardiac myosin characteristic rates of detachment and recruitment in the intact myofilament lattice are accelerated by Pi, phosphorylation of cMyBP-C, and the absence of cMyBP-C. The results suggest that cMyBP-C normally slows myosin detachment, including Pi-dependent detachment, and that this inhibition is released with phosphorylation or absence of cMyBP-C.