1. C-protein is a major component of muscle thick filaments whose function is unknown. We have examined for the first time the role of the regulatory binding domain of C-protein in modulating contraction and intracellular Ca2+ concentration ([Ca2+]i) in intact cardiac myocytes. 2. Rat ventricular myocytes were reversibly permeabilised with the pore-forming toxin streptolysin O. Myosin S2 (which binds to the regulatory domain of C-protein) was introduced into cells during permeabilisation to compete with the endogenous C-protein-thick filament interaction. 3. Introduction of S2 into myocytes increased contractility by approximately 30%, significantly lengthened the time to peak of the contraction and the time to half-relaxation, but had no effect on [Ca2+]i transient amplitude. 4. Our data are consistent with increased myofilament Ca2+ sensitivity when there is reduced binding of C-protein to myosin near the head-tail junction. 5. We propose that the effects of introducing S2 into intact cardiac cells can be equated with the consequences of selectively phosphorylating C-protein in vivo, and that the regulation of contraction by C-protein is mediated by the effects of crossbridge cycling on the Ca2+ affinity of troponin C.