Nitric oxide (NO.) inhibits mitochondrial respiration by binding to the binuclear heme α3/CuB center in cytochrome c oxidase. However, the significance of this reaction at physiological O2 levels (5-10 μM) and the effects of respiratory state are unknown. In this study mitochondrial respiration, absorption spectra, [O2], and [NO.] were measured simultaneously at physiological O2 levels with constant O2 delivery, to model in vivo respiratory dynamics. Under these conditions NO. inhibited mitochondrial respiration with an IC50 of 0.14 ± 0.01 μM in state 3 versus 0.31 ± 0.04 μM in state 4. Spectral data indicate that the higher sensitivity of state 3 respiration to NO. is due to greater control over respiration by an NO.-dependent spectral species in the respiratory chain in this state. These results are discussed in the context of regulation of respiration by NO. in vivo and its implications for the control of vessel-parenchymal O2 gradients.