We identified a novel mouse gene, mRTVP-1, as a p53 target gene using differential display PCR and extensive promoter analysis. The mRTVP-1 protein has 255 amino acids and differs from the human RTVP-1 (hRTVP-1) protein by two short in-frame deletions of two and nine amino acids. RTVP-1 mRNA was induced in multiple cancer cell lines by adenovirus-mediated delivery of p53 and by gamma irradiation or doxorubicin both in the presence and in the absence of endogenous p53. Analysis of RTVP-1 expression in nontransformed and transformed cells further supported p53-independent gene regulation. Using luciferase reporter and electrophoretic mobility shift assays we identified a p53 binding site within intron 1 of the mRTVP-1 gene. Overexpression of mRTVP-1 or hRTVP-1 induced apoptosis in multiple cancer cell lines including prostate cancer cell lines 148-1PA, 178-2BMA, PC-3, TSU-Pr1, and LNCaP, a human lung cancer cell line, H1299, and two isogenic human colon cancer cell lines, HCT116 p53(+/+) and HCT116 p53(-/-), as demonstrated by annexin V positivity, phase-contrast microscopy, and in selected cases 4',6'-diamidino-2-phenylindole staining and DNA fragmentation. Deletion of the signal peptide from the N terminus of RTVP-1 reduced its apoptotic activities, suggesting that a secreted and soluble form of RTVP-1 may mediate, in part, its proapoptotic activities.