p63, a recently identified member of the p53 gene family, encodes multiple products with transactivating, death-inducing, and dominant-negative activities. To explore the penetrance of p63 in bladder carcinogenesis, we performed expression and mutation analyses of two major isotypes, TAp63 and deltaNp63, in 63 bladder specimens. In 12 normal tissues, TAp63 was expressed at an easily detectable level whereas deltaNp63 was absent or extremely low. While none of 47 carcinomas showed allelic deletion of the gene, marked reduction of TAp63 and abnormal overexpression of deltaNp63 were found in 25 (53.2%) and 30 (63.8%) carcinomas, respectively. Tumor-specific alteration of TAp63 and deltaNp63 expression was identified in two and three of six matched sets, respectively. In addition, reduced expression of TAp63 showed a correlation with tumor stage and grade. Abnormal expression of TAp63 or deltaNp63 isoform was also observed in three of four cell lines, and treatment with 5-Aza-2'-deoxycytidine led to up- or down-regulation of TAp63 and/or deltaNp63 expression, suggesting that the promoters of both isoforms might be affected by DNA methylation, but not in a reciprocal fashion. No sequence alteration of p63 was identified in 47 carcinomas whereas 17 (34.8%) of these showed p53 mutations, and no association between p63 expression and the mutational status of p53 or expression of p21Waf1, MDM2, and 14-3-3sigma was recognized. Our data suggest that altered expression of p63 is a frequent event in bladder carcinogenesis and might contribute to the progression of bladder tumors, possibly via the mechanism(s) distinct from the p53 pathway.