Cancer cells are able to tolerate levels of O(2) that are damaging or lethal to normal cells; we hypothesize that this tolerance is the result of biochemical plasticity which maintains cellular homeostasis of both energy levels and oxidation state. In order to examine this hypothesis, we used different O(2) levels as a selective agent during long-term culture of DU145 prostate cancer cells to develop three isogenic cell lines that grow in normoxic (4%), hyperoxic (21%), or hypoxic (1%) O(2) conditions. Growth characteristics and O(2) consumption differed significantly between these cell lines without changes in ATP levels or altered sensitivity to 2-deoxy-D-glucose, an inhibitor of glycolysis. O(2) consumption was significantly higher in the hyperoxic line as was the level of endogenous superoxide. The hypoxic cell line regulated the chemical gradient of the proton motive force (PMF) independent of the electrical component without O(2)-dependent changes in Hif-1alpha levels. In contrast, the normoxic line regulated Hif-1alpha without tight regulation of the chemical component of the PMF noted in the hypoxic cell line. From these studies, we conclude that selection of prostate cancer cells by long-term exposure to low ambient levels of O(2) resulted in cells with unique biochemical properties in which energy metabolism, reactive oxygen species (ROS), and HIF-1alpha levels are modulated to allow cell survival and growth. Thus, cancer cells exhibit remarkable biochemical plasticity in response to various O(2) levels.