After the identification of p73, a second homologue of the human p53 tumor suppressor gene has been reported and named p63/p73L/p51/p40/CUSP/KET. We have investigated the hypotheses that: (a) p63 is mutated in diverse types of human cancers; and (b) p63 functions in the same pathway as p53 and p73 in the process of carcinogenesis; therefore, mutations in these three genes would be mutually exclusive. We have analyzed the genomic structure of the p63 gene and have performed mutational analyses on 54 human cell lines using intronic primers flanking each exon. We have confirmed that the human p63 open reading frame encodes the same length of protein as murine p63 that was initially reported to be 39 amino acids longer than human p63. By mutational analysis, we have shown that DLD1 and SKOV3 cells have either heterozygous mutations or polymorphisms in the putative DNA binding domain of p63. In these cell lines, p63 is biallelically expressed. We conclude that mutations in the p63 gene are rare in human cell lines. The fact that DLD1 is abnormal for both p63 and p53 genes suggests that they may not be involved in the same tumor suppressor pathway.