AbstractReactive oxygen species (ROS) and reactive nitrogen species (RNS) are usually identified with pathological states and mediators of cellular injury. However, over the last decade ROS and RNS have been identified in skeletal muscle under physiological conditions. Detection of ROS and RNS production by skeletal muscle cells is fundamental to the problem of differentiating between physiological and pathological levels. The goal of this paper is to review the techniques that have been used to detect ROS and RNS in skeletal muscle. Electron spin resonance, fluorescent assays, cyotchrome c reduction, chemiluminescence, hydroxylation of salicylate, and nitration of phenylalanine are some of the assay systems that have been used thus far. A large body of evidence now indicates that ROS and RNS are continually produced by many different skeletal muscle types studied in vivo, in situ, and in vitro. Under resting conditions, ROS and RNS are detectable in both intracellular and extracellular compartments. Production increases during both non‐fatiguing and fatiguing muscle contractions. In the absence of disease, the individual molecular species detected in skeletal muscle include parent radicals for the ROS and RNS cascades: superoxide anions and nitric oxide. Both are generated at rates estimated to range from pmol‐to‐nmol/mg muscle/minute. Evidence indicates that hydrogen peroxide, hydroxyl radicals, and peroxynitrite are also present under physiological conditions. However, the molecular species that mediate specific biological effects remains largely undetermined, as do the sources of ROS and RNS within muscle fibers. Eventual delineation of the mechanisms whereby ROS and RNS regulate cellular function will hinge on our understanding of the production and distribution of ROS and RNS within skeletal muscle. Microsc. Res. Tech. 55:236–248, 2001. © 2001 Wiley‐Liss, Inc.