To determine the role and mode of inactivation of the p16 and p15 genes in human esophageal tumors, we examined alterations and expression of the alpha and beta forms of the p16 gene, 5' CpG island methylation of p16 exon 1 alpha, and alterations of the p15 gene in 30 esophageal squamous-cell-carcinoma cell lines. Of 30 such cell lines examined, 28 (93%) showed aberrations of the alpha form of the p16 gene: 18 homozygous deletions, 6 point mutations and 4 hypermethylation. Methylation was exclusively observed in cell lines with the wild-type alpha form. Of the 6 point mutations, one was observed in exon 1 alpha, one in the splice acceptor site of intron 1 and the remaining 4 were in exon 2. In the beta form, 18 homozygous deletions and 3 point mutations in exon 2 were detected, but no point mutation was found in exon 1 beta. All mutations in exon 2 gave rise to premature termination codons in the reading frame of the alpha transcript, while no non-sense mutations were observed in the reading frame of the beta transcript. Among 12 cell lines without homozygous deletions of the alpha and beta forms of the p16 gene, the expected wild-type beta transcript was observed in 8 cell lines, whereas only one cell line expressed the expected wild-type alpha transcript. Homozygous deletions of the p15 gene were observed in 16 cell lines (53%), and no point mutations were detected. Twelve cell lines had alterations only in the alpha form of the p16 gene, while none showed aberrations exclusively in the p15 gene. Taken together, these results indicate that inactivation of the beta form of the p16 gene and the p15 gene are not so frequent as that of the alpha form of the p16 gene in ESC cell lines, suggesting that aberration of the alpha form of p16 gene is the primary target of 9p loss in ESC.