Mutational analysis of the p16/CDKN2 gene was conducted by direct sequencing of the whole coding sequence (exons 1-3 and flanking splicing sites) in 21 esophageal squamous-cell carcinomas and 3 adenocarcinomas from a high-incidence area of Italy. Two inactivating mutations were found in exon 1 of the gene (both in squamous-cell carcinoma), whereas no mutations were detected in exon 2, where most of the sequence changes reported so far have been located, or in exon 3. Southern blot analysis of exon 2 in this set of samples and in a complementary set of 12 tumor samples from France did not show homozygous deletions or detectable gene rearrangements. Thus, p16/CDKN2 gene alterations do not appear to play a major role in the group of patients examined.