In skeletal muscle, the functional role of uncoupling protein 3 (UCP3) in a physiological context is a point of serious contention in the field of mitochondrial physiology, due in part to the constraints imposed by the transgenic models (UCP3 knockouts display weak phenotypes, over-expressors display artifactual uncoupling) and the methodology used to investigate its function (in vitro assays of isolated mitochondria not representative of physiological state). A recently proposed theory suggests UCP3 acts as a natural antioxidant to reduce mitochondrial superoxide (O₂ ^•−) production. We developed an in situ technique to measure mitochondrial H₂O₂ emission (index of O₂^•−) in permeabilized rat skeletal myofibers to create a more physiologically relevant model of this process, and observed substantially more H₂O₂ coming from mitochondria in type II muscle fibers, of which red (RG) and white (WG) gastrocnemius are primarily composed, as compared to type I (soleus) muscle fibers. The low rate of mitochondrial H₂O₂ emission in soleus fiber bundles was shown to be a function of greater mitochondrial H₂O ₂ scavenging in these fibers, due partly to an increased activity of mitochondria glutathione peroxidase in the soleus fibers as compared to RG and WG. To investigate the function of UCP3 in a physiological context, mitochondrial H₂O₂ emission and respiration were measured in permeabilized fiber bundles prepared from rat RG and WG 18h after an acute bout of exercise (Ex/R) that induced a ∼2- to 4-fold increase in UCP3 protein compared to non-exercised controls. Elevated uncoupling activity (i.e., GDP sensitive) was evident only in Ex/R fibers, not in controls, upon addition of palmitate (known activator of UCP3) or under substrate conditions known to produce high rates of O₂^•− production (i.e., respiration supported by succinate or palmitoyl-L-carnitine/malate, but not pyruvate/malate), consistent with the purported activation of UCP3 by endogenous O₂ ^•−. This elevated uncoupling activity in Ex/R fibers was manifested in significantly lower rates of fatty acid-supported mitochondrial H₂O₂ emission in these fibers compared to controls. Collectively, these findings support the theory that UCP3 is an antioxidant feedback mechanism in skeletal muscle mitochondria, and provide a basis for consideration of in situ properties of mitochondria when studying mitochondrial oxidant production and UCP3 function in skeletal muscle.