The cardiac Ca2+ release channel [ryanodine receptor type 2 (RyR2)] is modulated by thiol reactive agents, but the molecular basis of RyR2 modulation by thiol reagents is poorly understood. Cys3635 in the skeletal muscle RyR1 is one of the most hyperreactive thiols and is important for the redox and calmodulin (CaM) regulation of the RyR1 channel. However, little is known about the role of the corresponding cysteine residue in RyR2 (Cys3602) in the function and regulation of the RyR2 channel. In the present study, we assessed the impact of mutating Cys3602 (C3602 A) on store overload-induced Ca2+ release (SOICR) and the regulation of RyR2 by thiol reagents and CaM. We found that the C3602 A mutation suppressed SOICR by raising the activation threshold and delayed the termination of Ca2+ release by reducing the termination threshold. As a result, C3602 A markedly increased the fractional Ca2+ release. Furthermore, the C3602 A mutation diminished the inhibitory effect of N-ethylmaleimide on Ca2+ release, but it had no effect on the stimulatory action of 4,4′- dithiodipyridine (DTDP) on Ca2+ release. In addition, Cys3602 mutations (C3602A or C3602R) did not abolish the effect of CaM on Ca2+-release termination. Therefore, RyR2-Cys3602 is a major site mediating the action of thiol alkylating agent N-ethylmaleimide, but not the action of the oxidant DTDP. Our data also indicate that residue Cys3602 plays an important role in the activation and termination of Ca2+ release, but it is not essential for CaM regulation of RyR2. © The Authors Journal compilation © 2015 Biochemical Society.