Oxidative phosphorylation not only generates cellular energy via ATP synthesis, but also controls the intracellular oxygen level to minimize oxygen toxicity resulting from reactive oxygen species (ROS). These species include superoxide (O2 -), hydrogen peroxide (H2O2), and hydroxyl radical (•OH). While the rate of mitochondrial respiration determines the intracellular oxygen concentration, the relationship between oxygen concentration and ROS generation is not fully understood. We hypothesized that mitochondrial respiration controls intracellular oxygen concentration which in turn regulates ROS generation. To test this hypothesis, we used two prostate cancer cell lines; PC-3 cells, which have low mitochondrial genome (mtDNA) content and low mitochondrial respiratory activity, and LNCaP cells, which have high mtDNA content and high mitochondrial respiratory activity. PC-3 cells exhibited high mitochondrial oxygen concentration and generated more O2 - as well as •OH when compared to LNCaP cells which showed low mitochondrial oxygen concentration and reduced levels of O2 - and •OH. Exogenous hypoxic conditions (0.2% O2) reduced mitochondrial oxygen concentration and the levels of ROS, whereas exogenous hyperoxic conditions (40% O2) increased mitochondrial oxygen concentration and increased the levels of ROS. These results support the hypothesis that mitochondrial respiration regulates the intracellular oxygen concentration and in turn the generation of ROS.