We have shown previously that at physiologically relevant oxygen tension (pO(2) approximately 10 mmHg), NO S-nitrosylates 1 of approximately 50 free cysteines per ryanodine receptor 1 (RyR1) subunit and transduces a calcium-sensitizing effect on the channel by means of calmodulin (CaM). It has been suggested that cysteine-3635 is part of a CaM-binding domain, and its reactivity is attenuated by CaM [Porter Moore, C., Zhang, J. Z., Hamilton, S. L. (1999) J. Biol. Chem. 274, 36831-36834]. Therefore, we tested the hypothesis that the effect of NO was mediated by C3635. The full-length RyR1 single-site C3635A mutant was generated and expressed in HEK293 cells. The mutation resulted in the loss of CaM-dependent NO modulation of channel activity and reduced S-nitrosylation by NO to background levels but did not affect NO-independent channel modulation by CaM or the redox sensitivity of the channel to O(2) and glutathione. Our results reveal that different cysteines within the channel have been adapted to serve in nitrosative and oxidative responses, and that S-nitrosylation of the cysteine-containing CaM-binding domain underlies the mechanism of CaM-dependent regulation of RyR1 by NO.