Proline oxidase is a p53-induced redox gene that can generate reactive oxygen species (ROS) and mediate apoptosis in tumor cells. We report that proline oxidase is a downstream effector in p53-mediated activation of the calcium/calmodulin-dependent phosphatase calcineurin in lung, renal, colon, and ovarian carcinoma cells. The activation of calcineurin by p53 and proline oxidase was detected by activation of the nuclear factor of activated T cells (NFAT), an established indicator of activated calcineurin. Both proline oxidase- and p53-induced activation of NFAT were sensitive to the calcineurin inhibitors cyclosporin A and FK-506, to scavengers of ROS, and to inhibitors of calcium mobilization. A proline oxidase antisense vector suppressed the ability of p53 to up-regulate proline oxidase, activate calcineurin, and induce apoptosis. Moreover, two renal carcinoma-derived mutant p53 proteins were deficient in inducing proline oxidase expression and in activating calcineurin. Inhibitors of calcineurin and calcium mobilization abolished proline oxidase-mediated apoptosis and reduced p53-induced apoptosis. Treatment of colon and ovarian carcinoma cells with the anticancer genotoxic agent etoposide up-regulated both p53 and proline oxidase, activated calcineurin, and induced apoptosis. The etoposide-mediated activation of calcineurin and induction of apoptosis was markedly suppressed by FK-506 calcineurin inhibitor. We propose that proline oxidase mediates apoptosis through the generation of proline-dependent ROS, which then mobilize calcium and activate calcineurin. The activation of calcineurin-regulated transcription factor pathways by proline oxidase might affect gene expression events important to p53 regulation of cell growth and apoptosis.