We evaluated the clinicopathologic significance of p53 gene mutations, including a comparison of DNA analysis and immunohistochemical examination, in Japanese patients with esophageal squamous cell carcinoma, a highly aggressive cancer. Genomic DNA isolated from 76 tumors without preoperative treatment was subjected to polymerase chain reaction and sequencing. Associations were sought between p53 mutations and clinicopathologic characteristics. Cases also were investigated immunohistochemically to detect abnormal p53 protein accumulation. Overexpression of p53 protein occurred in 51 cases (67.1%), while gene mutations in the examined exons were found in only 14 (18.4%). By multivariate analysis, p53 mutation predicted detection of eight or more lymph node metastases. Mutations of the p53 gene may not only participate in the initiation of esophageal cancer, but also may promote lymph node metastasis. Unlike gene mutations, p53 protein overexpression did not predict nodal metastasis extent.