More than half of human cancers contain mutations in the tumor suppressor protein p53, most of which accumulate in the DNA binding domain of the protein. Here we report the identification of a mutant p53, designated p53-46F, in which Ser-46 is replaced with phenylalanine. In vitro, adenovirus-mediated transduction of the p53-46F gene induced apoptosis more efficiently than wild-type p53 in a number of cancer cell lines, whereas Ser-15 phosphorylation of p53-46F was enhanced in all cancer cell lines examined. Moreover, the expression level of the cell cycle inhibitor p21/WAF1 was decreased in cell lines infected with adenovirus p53-46F (Ad-p53-46F). p53-46F caused a more enhanced level of transcriptional activation of several p53-target genes, including Noxa, p53AIP1 and p53RFP, compared with wild-type p53. In vivo, adenovirus-mediated gene transfer of p53-46F enhanced apoptosis, thus suppressing tumor growth of a lung cancer cell line more effectively than wild-type p53 or p53-121F, another p53 mutant. Collectively, our data suggest that p53-46F is an active version of p53 that demonstrates enhanced induction of p53-dependent apoptosis. This is probably mediated by upregulated transactivation of genes downstream of p53, increased Ser-15 phosphorylation and a decrease in p21/WAF1 levels. We propose p53-46F as an alternative candidate to wild-type p53 for use in developing new therapeutic strategies for the treatment of cancer.