Our previous studies of esophageal squamous cell carcinoma (ESC) revealed that frequent loss of heterozygosity (LOH) on 13q12-13 was associated with lymph node metastasis. These results suggest that 13q12-13 may harbor a tumor suppressor gene which regulates lymph node metastasis. Recently, a new tumor suppressor candidate, AS3, was identified in this region. We therefore examined AS3 alterations by PCR-SSCP and RT-PCR. Although polymorphisms were found in exon 10 and introns 21, 23, 25 and 29, a possible tumor specific mutation in exon 6 was identified in only one of 31 ESC-derived cell lines, indicating that the remaining allele of AS3 was rarely inactivated by mutational events. Interestingly, ESC patients with rare alleles frequently exhibited lymph node metastasis. These results suggest that polymorphic variation and LOH at the AS3 locus might be associated with lymph node involvement in ESC.