Reactive disulfide compounds (RDSs) with a pyridyl ring adjacent to a disulfide bond, 2,2′dithiodipyridine (2,2′ DTDP) and 4,4′ dithiodipyridine (4,4′ DTDP), induce Ca2+ release from isolated canine cardiac sarcoplasmic reticulum (SR) vesicles. RDSs are absolutely specific to free sulfhydryl (SH) groups and oxidize SH sites of low pKa via a thiol-disulfide exchange reaction, with the stoichiometric production of thiopyridone in the medium. As in skeletal SR, this reaction caused large increases in the Ca2+ permeability of cardiac SR and the number of SH sites oxidized by RDSs was kinetically and quantitatively measured through the absorption o f thiopyridone. RDS-induced Ca2+ release from cardiac SR was characterized and compared to the action of RDSs on skeletal SR and to Ca2+-induced Ca2+ release, (i) RDS-induced Ca2+ release from cardiac SR was dependent on ionized Mg2+, with maximum rates of release occurring at 0.5 and 1 mm Mgfree2+ for 2,2′ DTDP and 4,4′ DTDP, respectively, (ii) In the presence of adenine nucleotides (0.1-1 mm), the oxidation of SH sites in cardiac SR by exogenously added RDS was inhibited, which, in turn, inhibited Ca2+ release induced by RDSs. (iii) Conversely, when the oxidation reaction between RDSs and cardiac SR was completed and Ca2+ release pathways were opened, subsequent additions of adenine nucleotides stimulated Ca2+ efflux induced by RDSs. (iv) Sulfhydryl reducing agents (e.g., dithiothreitol, DTT, 1-5 mm) inhibited RDS-induced Ca2+ efflux in a concentration-dependent manner, (v) RDSs elicited Ca2+ efflux from passively loaded cardiac SR vesicles (i.e., with nonfunctional Ca2+ pumps in the absence of Mg-ATP) and stimulated Ca2+-dependent ATPase activity, which indicated that RDS uncoupled Ca2+ uptake and did not act at the Ca2+, Mg2+-ATPase. These results indicate that RDSs selectively oxidize critical sulfhydryl site(s) on or adjacent to a Ca2+ release channel protein channel and thereby trigger Ca2+ release. Conversely, reduction of these sites reverses the effects of RDSs by closing Ca2+ release channels, which results in active Ca2+ reuptake by Ca2+, Mg2+-ATPase. These compounds can thus provide a method to covalently label and identify the protein involved in Ca2+ release from cardiac SR.