Experimental evidence is presented demonstrating the existence of a potent O2 . - 2 source in heart mitochondria. The novel O . - 2 generator is more active than any other known mitochondrial O . - 2 generator and also exhibits a higher affinity for molecular oxygen. In contrast to mitochondrial O . - 2 sources reported previously [(1974) FEBS Lett. 42, 68-72; (1978) Eur. J. Biochem. 82, 563-567], the O . - 2 generator described in this paper is not involved in energy-linked respiration. Superoxide radicals from this source require NADH to initiate their generation, and the radicals formed are released entirely into the extramitochondrial space. NADH-related O . - 2 generation was also observed with the solubilized erogenous NADH oxidoreductase of heart mitochondria, an enzyme recently described [(1987) Eur. J. Biochem., submitted]. This finding together with the lack of an NADH-dependent O . - 2 source in liver mithochondria suggests that the novel O . - 2 generator and the erogenous NADH oxidoreductase of heart mitochondria are identical.