Superoxide (O(2)(-)) is the proximal mitochondrial reactive oxygen species underlying pathology and redox signaling. This central role prioritizes development ofa mitochondria-targeted reagent selective for controlling O(2)(-). We have conjugated a mitochondria-targeting triphenylphosphonium (TPP) cation to a O(2)(-)-selective pentaaza macrocyclic Mn(II) superoxide dismutase (SOD) mimetic to make MitoSOD, a mitochondria-targeted SOD mimetic. MitoSOD showed rapid and extensive membrane potential-dependent uptake into mitochondria without loss ofMn and retained SOD activity. Pulse radiolysis measurements confirmed that MitoSOD wasa veryeffective catalytic SOD mimetic. MitoSOD also catalyzes the ascorbate-dependent reduction of O(2)(-). The combination of mitochondrial uptake and O(2)(-) scavenging by MitoSOD decreased inactivation of the matrix enzyme aconitase caused by O(2)(-). MitoSOD is an effective mitochondria-targeted macrocyclic SOD mimetic that selectively protects mitochondria from O(2)(-) damage.