The oxygen dependence of mitochondrial oxidative phosphorylation was measured in suspensions of isolated rat liver mitochondria using recently developed methods for measuring oxygen and cytochrome c reduction. Cytochrome-c oxidase (energy conservation site 3) activity of the mitochondrial respiratory chain was measured using an artificial electron donor (N,N,N',N'-tetramethyl-p-phenylenediamine) and ascorbate to directly reduce the cytochrome c, bypassing sites 1 and 2. For mitochondrial suspensions with added ATP, metabolic conditions approximating those in intact cells and decreasing oxygen pressure both increased reduction of cytochrome c and decreased respiratory rate. The kinetic parameters [K(M) and maximal rate (V(M))] for oxygen were determined from the respiratory rates calculated for 100% reduction of cytochrome c. At 22°C, the K(M) for oxygen is near 3 Torr (5 μM), 12 Torr (22 μM), and 18 Torr (32 μM) at pH 6.9, 7.4, and 7.9, respectively, and V(M) corresponds to a turnover number for cytochrome c at 100% reduction of near 80/s and is independent of pH. Uncoupling oxidative phosphorylation increased the respiratory rate at saturating oxygen pressures by twofold and decreased the K(M) for oxygen to <2 Torr at all tested pH values. Mitochondrial oxidative phosphorylation is an important oxygen sensor for regulation of metabolism, nutrient delivery to tissues, and cardiopulmonary function. The decrease in K(M) for oxygen with acidification of the cellular environment impacts many tissue functions and may give transformed cells a significant survival advantage over normal cells at low-pH, oxygen-limited environment in growing tumors.