Mammalian skeletal muscles generate marked amounts of superoxide (O2·−) at 37°C, but it is not well understood which is the main source of O2·− production in the muscle fibers and how this interferes with muscle function. To answer these questions, O2·− production and twitch force responses were measured at 37°C in mechanically skinned muscle fibers of rat extensor digitorum longus (EDL) muscle. In mechanically skinned fibers, the sarcolemma is removed avoiding potential sources of O2·− production that are not intrinsically part of the muscle fibers, such as nerve terminals, blood cells, capillaries and other blood vessels in the whole muscle. O2·− production was also measured in split single EDL muscle fibers, where part of the sarcolemma remained attached, and small bundles of intact isolated EDL muscle fibers at rest, in the presence and absence of modifiers of mitochondrial function. The results lead to the conclusion that mitochondrial production of O2·− accounts for most of the O2·− measured intracellularly or extracellularly in skeletal muscle fibers at rest and at 37°C. Muscle fiber excitability at 37°C was greatly improved in the presence of a membrane permeant O2·− dismutase mimetic (Tempol), demonstrating a direct link between O2·− production in the mitochondria and muscle fiber performance. This implicates mitochondrial O2·− production in the down-regulation of skeletal muscle function, thus providing a feedback pathway for communication between mitochondria and plasma membranes that is not directly related to the main function of mitochondria as the power plant of the mammalian muscle cell.