Two unresolved aspects of the role of mitochondrial-derived cytochrome c in apoptosis are whether there is a separate pool of cytochrome c within mitochondria that participates in the activation of apoptosis and whether a chemically modified cytochrome c drives apoptosis. These questions were investigated using osteoclasts since they are rich in mitochondria and osteoclast apoptosis is critical in bone metabolism regulation. Hydrogen peroxide production was increased during culture, preceding cytochrome c release; both processes occurred anterior to apoptosis. By adding a mitochondrial uncoupler, hydrogen peroxide production and apoptosis were blocked indicating the prominent role of mitochondrial-derived hydrogen peroxide. Trapping hydrogen peroxide-derived hydroxyl radical decreased apoptosis. Cytosolic cytochrome c was originated from a single mitochondrial compartment, supporting a common pool involved in respiration and apoptosis and it was chemically identical to the native form, with no indication of oxidative or nitrative modifications. Protein levels of Bcl-2 and BcxL were decreased prior to apoptosis, whereas expressing wild type Bcl-2 repressed apoptosis, confirming that cytochrome c release is critical to initiating apoptosis. Cytosolic cytochrome c participated at activating caspases 3 and 9, both being required for apoptosis. Collectively, our data indicate that the mitochondrial dependent apoptotic pathway is one of the major routes operating in osteoclasts.