Superoxide released from mitochondria forms reactive oxygen species that can cause severe oxidative damage and have been associated with aging- and disuse-induced muscle dysfunction. Superoxide is released to both the exterior and the matrix of mitochondria, where oxidative damage is not necessarily the same. This complicates determining the role of mitochondrial superoxide in eliciting oxidative stress in skeletal muscle. A newly developed capillary electrophoretic method analyzes hydroxytriphenylphosphonium ethidium, a superoxide-specific product of triphenylphosphonium hydroethidine, released to outside the mitochondria (supernatant) and retained in the matrix (pellet). In this study, we investigated the mitochondrial superoxide production of soleus (type I) and semimembranosus (type II) muscles of Fischer 344 rats affected by aging (13 vs. 26 mo) and disuse (hindlimb unloading). In agreement with previous studies, overall superoxide production increased with aging and disuse. On the other hand, the new experimental method revealed that superoxide production outside the mitochondria of the soleus does not show a significant age-related increase. Another observation was that the superoxide production increase in the matrix occurs earlier (7 days of disuse) compared with the outside mitochondria (14 days of disuse) in both muscle types. These findings indicate that superoxide release is complex as it occurs asymmetrically at both sides of the mitochondrial inner membrane, and that such release has muscle type and temporal specificity. These findings are important to refine current concepts on oxidative stress associated with muscle aging and disuse.