Ion channels have been studied extensively in ambient O₂ tension (pO₂), whereas tissue pO₂ is much lower. The skeletal muscle calcium release channel/ryanodine receptor (RyR1) is one prominent example. Here we report that pO₂ dynamically controls the redox state of 6–8 out of 50 thiols in each RyR1 subunit and thereby tunes the response to NO. At physiological pO₂, nanomolar NO activates the channel by S-nitrosylating a single cysteine residue. Among sarcoplasmic reticulum proteins, S-nitrosylation is specific to RyR1 and its effect on the channel is calmodulin dependent. Neither activation nor S-nitrosylation of the channel occurs at ambient pO₂. The demonstration that channel cysteine residues subserve coupled O₂ sensor and NO regulatory functions and that these operate through the prototypic allosteric effector calmodulin may have general implications for the regulation of redox-related systems.