Although the production of reactive oxygen species (ROS) during muscle contractile activity has been linked to both positive and negative adaptive responses, the sites for ROS generation within working muscle are not clearly defined. We assessed cytosolic ROS production and mitochondrial redox potential with a targeted redox-sensitive green fluorescent protein during repetitive field stimulation of single mature myofibers. Cytosolic ROS production increased by 94%, an effect that was abolished by pretreatment with the reducing agent dithiothreitol. Mitochondrial redox potential was not altered during muscle contraction. In contrast, activity-dependent ROS production was ablated by an inhibitor of NADPH oxidase. We provide the first report on dynamic ROS production from mitochondria in single living myofibers and suggest that the mitochondria are not the major source of ROS during skeletal muscle contraction. Alternatively, our data support a role for NADPH oxidase-derived ROS during contractile activity.