Mutations in Cu, Zn superoxide dismutase (SOD1) cause a fraction of amyotrophic lateral sclerosis (ALS), which involves motoneuron degeneration, paralysis, and death. An acquired activity by mutant SOD1 is responsible for the cellular toxicity, but how mutant SOD1 kills motoneurons is unclear. In transgenic mouse models of ALS, mitochondrial degeneration occurs early, before disease onset, raising the question of how mutant SOD1 damages mitochondria. Here we investigate the intracellular localization of SOD1 in the CNS to determine whether SOD1 is present in mitochondria, where it could directly damage this organelle. We show that endogenous mouse SOD1, wild-type human, and mutant human SOD1 (G93A), when expressed as transgenes, are colocalized with mitochondria in spinal cord by immunofluorescence confocal microscopy. By immunoelectron microscopy, we show that SOD1 is present within mitochondria at similar concentrations as in the cytoplasm. Thus SOD1, in addition to being a cytosolic enzyme, is present inside mitochondria in the CNS.