Binding of nitric oxide (NO) to isolated cytochrome c oxidase in turnover was investigated by static and kinetic spectroscopic methods. These studies indicate that cytochrome c oxidase rapidly binds NO when the enzyme enters turnover. Our results show that NO binds to ferrocytochrome a3, competing with oxygen for this binding site. However, the main features of the binding process, in particular the rapid onset of inhibition, cannot be fully explained on this basis. We suggest, therefore, that there is a second binding site for NO, which has lower affinity but nevertheless plays an important role in the inhibitory process. A likely possibility is that Cu(B)+ constitutes this second binding site. The fast onset of inhibition observed in the presence of NO, along with the dependence on the oxygen concentration, suggests that under physiological conditions, where the oxygen concentration is low, nanomolar concentrations of NO can effectively act as a regulator of the mitochondrial respiratory chain.