Most cultured cells and intact animals die under hyperoxic conditions. However, a strain of HeLa cells that proliferates under 80% O2, termed "HeLa-80," has been derived from wildtype HeLa cells ("HeLa-20") by selection for resistance to stepwise increases of oxygen partial pressure. The tolerance of HeLa-80 cells to hyperoxia is not associated with changes in antioxidant defenses or susceptibility to oxidant-mediated killing. Rather, under both 20 and 80% O2, mitochondrial reactive oxygen species (ROS) production is ∼2-fold less in HeLa-80 cells, likely related to a significantly higher cytochrome c oxidase (COX) activity (∼2-fold), which may act to deplete upstream electron-rich intermediates responsible for ROS generation. We now report that in HeLa-80 cells elevated COX activity is associated with a >2-fold increase in the regulatory subunit COX Vb, whereas expression levels of other subunits are very close to wild type. Small interfering RNA against Vb selectively lowers COX Vb expression in HeLa-80 cells, increases mitochondrial ROS generation, decreases COX activity 60-80%, and diminishes viability under 80% (but not 20%) O 2. In addition, overexpression of subunit Vb increases COX activity and decreases ROS production in wild-type HeLa-20 cells, along with some increase in tolerance to hyperoxia. Overall, our results indicate that it is possible to make cells tolerant of hyperoxia by manipulation of mitochondrial electron transport. These observations may suggest new pharmaceutical strategies to diminish oxygen-mediated cellular damage.