The effect of the free radical nitric oxide (NO) on the activity of isolated cytochrome c oxidase was investigated by using ferrocytochrome c as an electron donor, and the system SNOG/DTT, which produces a steady-state NO concentration similar to that expected to be found in vivo. The initial electron entry into the heme a/Cu(a) center and the initial rate of the electron transfer between the two hemes were not affected by the presence of NO. Under our conditions, the rate of inhibition of cytochrome c oxidase was found to be dependent both on the SNOG (NO concentration) and on the ferrocytochrome c concentration (electron entry rate). The data confirm that NO binds exclusively at the binuclear center, and that the NO binding in these conditions requires the presence of an intermediate populated only in turnover. Accordingly, we found that the rate of inhibition is directly related to the electron entry rate. In addition, a residual activity seems to be present in cytochrome c oxidase in the presence of nitric oxide, suggesting that NO can act as an electron acceptor to cytochrome c oxidase in the presence of oxygen.