Fischer-344 rats were exposed for 4 hr to various concentrations of hydrogen sulfide (H2S) gas and killed either immediately or at 1, 24, or 48 hr after exposure. Mitochondrial fractions from lung tissues were assayed for the activities of respiratory chain enzymes. Exposure of rats to a low concentration (10 ppm) of H2S caused no significant changes in the activities of lung mitochondrial enzymes. However, exposure to sublethal concentrations of H2S (50-400 ppm) produced marked and highly significant depressions in the activities of cytochrome c oxidase and succinate oxidase complexes of the respiratory chain. The inhibition of cytochrome c oxidase activity in lungs was most severe (greater than 90%) in rats that died from acute exposure to greater than 500 ppm H2S. In rats exposed to 200 and 400 ppm H2S, a marked recovery in cytochrome c oxidase activity of lungs was observed at 24 and 48 hr postexposure. Studies in vitro with rat lung mitochondria showed that low concentrations of sulfide also caused a similar and selective inhibition of cytochrome c oxidase activity. This effect was reversed upon removal of sulfide either by washing or by oxidation with methemoglobin. The nature of sulfide inhibition of cytochrome c oxidase was noncompetitive with respect to ferrocytochrome c. Because the activities of NADH-cytochrome c reductase and succinate-cytochrome c reductase were not significantly altered by H2S exposure and in vitro treatments with low concentrations of sulfide, it is concluded that under physiological conditions H2S would block the respiratory chain primarily by inhibiting cytochrome c oxidase. Such a biochemical impairment would lead to functional (histotoxic) hypoxia in the lung tissues.