Biological systems that produce or are exposed to nitric oxide (NO .) exhibit changes in the rate of oxygen free radical production. Considering that mitochondria are the main intracellular source of oxygen radicals, and based on the recently documented production of NO. by intact mitochondria, we investigated whether NO., produced by the mitochondrial nitric-oxide synthase, could affect the generation of oxygen radicals. Toward this end, changes in H2O2 production by rat liver mitochondria were monitored at different rates of endogenous NO . production. The observed changes in H202 production indicated that NO. affected the rate of oxygen radical production by modulating the rate of O2 consumption at the cytochrome oxidase level. This mechanism was supported by these three experimental proofs: 1) the reciprocal correlation between H2O 2 production and respiratory rates under different conditions of NO. production; 2) the pattern of oxidized/re duced carriers in the presence of NO., which pointed to cytochrome oxidase as the crossover point; and 3) the reversibility of these effects, evidenced in the presence of oxymyoglobin, which excluded a significant role for other NO .-derived species such as peroxynitrite. Other sources of H 2O2 investigated, such as the aerobic formation of nitrosoglutathione and the GSH-mediated decay of nitrosoglutathione, were found quantitatively negligible compared with the total rate of H2O 2 production.