Myosin binding protein-C (MyBP-C) is a poorly understood component of the thick filament in striated muscle sarcomeres. Its C terminus binds tightly to myosin, whereas the N terminus contains binding sites for myosin S2 and possibly for the thin filament. To study the role of the N-terminal domains of cardiac MyBP-C (cMyBP-C), we added human N-terminal peptide fragments to human and rodent skinned ventricular myocytes. At concentrations >10 μmol/L, the N-terminal C0C2 peptide activated force production in the absence of calcium (pCa 9). Force at the optimal concentration (80 μmol/L) of C0C2 was ≈60% of that in maximal Ca²⁺ (pCa 4.5), but the rate constant of tension redevelopment (k_tr) matched or exceeded (by up to 80%) that produced by Ca²⁺ alone. Experiments using different N-terminal peptides suggested that this activating effect of C0C2 resulted from binding by the pro/ala-rich C0-C1 linker region, rather than the terminal C0 domain. At a lower concentration (1 μmol/L), exogenous C0C2 strongly sensitized cardiac myofibrils to Ca²⁺ at a sarcomere length (SL) of 1.9 μm but had no significant effect at SL 2.3 μm. This differential effect caused the normal SL dependence of myofibrillar Ca²⁺ sensitivity to be reduced by 80% (mouse myocytes) or abolished (human myocytes) in 1 μmol/L C0C2. These results suggest that cMyBP-C provides a regulatory pathway by which the thick filament can influence the activation of the thin filament, separately from its regulation by Ca²⁺. Furthermore, the N-terminal region of cMyBP-C can influence the SL-tension (Frank–Starling) relationship in cardiac muscle.