Homozygous and heterozygous deletions involving chromosome 9p21 have been reported in a variety of primary human tumors in vivo, and point mutations have been reported in melanoma cell lines in vitro within a probable tumor suppressor gene, MTS1, located at chromosome 9p21. We describe six sequence alterations occurring among twenty-four primary esophageal squamous carcinomas and nineteen primary esophageal adenocarcinomas analyzed by DNA sequencing of MTS1 exon 2. Nucleotide substitutions were observed in five squamous cell carcinomas and in one adenocarcinoma. Two occurred in the germline, while four were somatic alterations. All six nucleotide changes resulted in marked alterations in amino acid sequence. Four were nonsense mutations leading to premature termination codons; nucleotide substitutions identical to two of these stop codons were previously reported in other tumor types. Loss of heterozygosity occurred in all five informative (constitutionally heterozygous) cases in which a sequence alteration was present. Esophageal cancer is one primary human tumor in which MTS1 constitutes an apparent target of heterozygous or homozygous deletions occurring at chromosome 9p21.