The manganese superoxide dismutase (Mn-SOD) converts superoxide anions to hydrogen peroxide plus oxygen, providing the first line of defense against oxidative stress in mitochondria. Heart mitochondria exhibited higher Mn-SOD activity than liver mitochondria. In mitochondria from both tissues Mn-SOD activity decreased after incubation at low oxygen concentration (hypoxic mitochondria). The effects of free Ca(2+) ([Ca(2+)](f)) and free Mg(2+) ([Mg(2+)](f)) on normoxic and hypoxic mitochondria from either organ were tested. In normoxic mitochondria from either tissue, both [Ca(2+)](f) and [Mg(2+)](f) activated the enzyme, although [Mg(2+)](f) was less efficient as an activator and the effect was lower in heart than in liver mitochondria. When added simultaneously, high [Ca(2+)](f) and [Mg(2+)](f) exhibited additive effects which were more pronounced in heart mitochondria and were observed regardless of whether mitochondria had been incubated under normal or low oxygen. The data suggest that [Ca(2+)](f) plays a role in regulating Mn-SOD in concert with the activation of aerobic metabolism.