Physiological sensing of O(2) tension (partial O(2) pressure, pO(2)) plays an important role in some mammalian cellular systems, but striated muscle generally is not considered to be among them. Here we describe a molecular mechanism in skeletal muscle that acutely couples changes in pO(2) to altered calcium release through the ryanodine receptor-Ca(2+)-release channel (RyR1). Reactive oxygen species are generated in proportion to pO(2) by NADPH oxidase 4 (Nox4) in the sarcoplasmic reticulum, and the consequent oxidation of a small set of RyR1 cysteine thiols results in increased RyR1 activity and Ca(2+) release in isolated sarcoplasmic reticulum and in cultured myofibers and enhanced contractility of intact muscle. Thus, Nox4 is an O(2) sensor in skeletal muscle, and O(2)-coupled hydrogen peroxide production by Nox4 governs the redox state of regulatory RyR1 thiols and thereby governs muscle performance. These findings reveal a molecular mechanism for O(2)-based signaling by an NADPH oxidase and demonstrate a physiological role for oxidative modification of RyR1.