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Abstract-The role of cardiac myosin-binding protein C (cMyBP-C) in cardiac contraction is still not fully resolved.Experimental ablation of cMyBP-C by various means resulted in inconsistent changes in Ca 2ϩ sensitivity and increased velocity of force of skinned preparations. To evaluate how these effects are integrated in an intact, living myocyte context, we investigated consequences of cMyBP-C ablation in ventricular myocytes and left atria from cMyBP-C knock-out (KO) mice compared with wild-type (WT). At 6 weeks, KO myocytes exhibited mild hypertrophy that became more pronounced by 30 weeks. Isolated cells from KO exhibited markedly lower diastolic sarcomere length (SL) without change in diastolic Ca 2ϩ . The lower SL in KO was partly abolished by the actin-myosin ATPase inhibitors 2,3-butanedione monoxime or blebbistatin, indicating residual actin-myosin interaction in diastole. The relationship between cytosolic Ca 2ϩ and SL showed that KO cells started to contract at lower Ca 2ϩ without reaching a higher maximum, yielding a smaller area of the phase-plane diagram. Both sarcomere shortening and Ca 2ϩ transient were prolonged in KO. Isolated KO left atria exhibited a marked increase in sensitivity to external Ca 2ϩ and, in contrast to WT, continued to develop twitch force at low micromolar Ca 2ϩ . Taken together, the main consequence of cMyBP-C ablation was a defect in diastolic relaxation and a smaller dynamic range of cell shortening, both of which likely result from the increased myofilament Ca 2ϩ sensitivity. Our findings indicate that cMyBP-C functions as a restraint on myosin-actin interaction at low Ca 2ϩ and short SL to allow complete relaxation during diastole. (Circ Res. 2007;101:928-938.)