Reactive oxygen species (ROS) play an important role in normal signaling events and excessive ROS are associated with many pathological conditions. The amount of ROS in cells is dependent on both the production of ROS by the mitochondrial electron transport chain and their removal by ROS-detoxifying enzymes. The peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) is a master regulator of mitochondrial functions and a key regulator of the ROS-detoxifying program. However, the impact of PGC-1α on the topology and rate of superoxide production by the mitochondrial electron transport chain is not known. We report here, using mitochondria from muscle creatine kinase–PGC-1α transgenic mice, that PGC-1α does not affect the topology of ROS production, but increases the capacity of complexes I and III to generate ROS. These changes are associated with increased mitochondrial respiration and content of respiratory chain complexes. When normalizing ROS production to mitochondrial respiration, we find that PGC-1α preserves the percentage of free radical leak by the electron transport chain. Together, these data demonstrate that PGC-1α regulates the intrinsic properties of mitochondria in such a way as to preserve a tight coupling between mitochondrial respiration and ROS production.