An increased production of oxygen radicals has been postulated to be a major factor in the etiology of lung damage during hyperoxia. Mitochondrial electron transport was inhibited with CN-or with antimycin A in both rat lung slices and isolated mitochondria. CN” or antimycin A-insensitive O2 uptake was measured polarographically, as a function of PO,, and served as an approximate index of intracellular 02-and Hz02 production. In lung slices, CN--resistant respiration increased as a function of Po,, accounting for 9% of total respiration in air and becoming 18% of total respiration when the tissue was incubated in 85% 02. CN--resistant respiration in isolated mitochondria also increased as a function of Po2 rising from 0 at 15% O2 to 1.34 nmol of O2 consumed/min* mg of mitochondrial protein at 85% 02. Mitochondria accounted for 15 f: 3% of the CN” resistant respiration in rat lungs under hyperoxic conditions and released H202 extramitochondrially at a rate of 50 nmol/min/l. 5 g of rat lung. The HzO2 generation is dependent on Po, and substrate and most, if not all, Hz02 arises from dismutation of 02-produced by autooxidation of respiratory chain components. 2, 4-Dinitrophenol increased respiratory chain 02-production in a dose-dependent fashion. This phenomenon occurred when mitochondria were treated with nitroaromatic compounds which can be reduced to nitroanion free radicals capable of reducing O2 to 02-. Nonreducible uncouplers such as salicylate did not increase mitochondrial 02-generation, suggesting that uncoupling, per se, does not necessarily favor increased rates of mitochondrial 02-production. These data suggest that hyperoxia increases the pulmonary production of oxygen radicals and that mitochondria contribute sig-nificantly to this phenomenon.