The p53-dependent initiation of apoptosis is accompanied by the induction of proline oxidase (POX), a mitochondrial enzyme catalyzing the conversion of proline to pyrroline-5-carboxylate with the concomitant transfer of electrons to cytochrome c. However, the contribution of increased POX activity to apoptosis, if any, remains unknown. Using Adriamycin to initiate p53-dependent apoptosis, we showed that the expression of POX is up-regulated in a time- and dose-dependent manner in a human colon cancer cell line (LoVo). In cells expressing POX, the addition of proline increases reactive oxygen species (ROS) generation in a concentration-dependent manner; glutamate, a downstream product of proline oxidation, had no effect. Induction of POX was dependent on the p53 status of the cell. In the conditionally immortalized murine colonic epithelial cell line YAMC, where the p53 phenotype can be modulated by temperature, proline oxidase expression and ROS production could only be induced when the cells were phenotypically p53-positive. To confirm that the observed ROS production was not secondary to some other effect of p53, we also conditionally expressed POX in a p53-negative colon cancer line. Again, we found a proline-dependent ROS increase with POX expression. We hypothesize that proline oxidation supports the generation of ROS by donating reducing potential to an electron transport chain altered either by p53-dependent mechanisms or by overexpression of POX.