Troponin C regulates contraction in striated muscle by alternating between the Ca(2+)-bound and apo conformations. We report here that spontaneous formation of an intramolecular disulfide bond between Cys-35 and Cys-84, or dimerization via an intermolecular disulfide bond between Cys-84 in cardiac troponin C, renders the protein Ca(2+)-independent when assayed in fast skeletal muscle myofibrils but to a much lesser extent in cardiac myofibrils. Formation of the intramolecular disulfide bond appears to expose hydrophobic surfaces, as indicated by an increase in fluorescence from hydrophobic fluorescent dyes, but does not alter the affinity of Ca(2+)-binding site II. These disulfide bonds constrain the protein into a conformation that either resembles or can substitute for the Ca(2+)-bound form of cardiac troponin C in fast skeletal muscle myofibrils.