In esophageal squamous cell carcinoma, p53 gene mutations have been analyzed for inter- or intra-patient heterogeneity but only a few studies have investigated intratumoral heterogeneity. We investigated this question within individual esophageal cancers, and also in their lymph-node metastases in 8 cases. Analyzing the p53 gene sequence by direct sequencing of polymerase chain reaction products, we found heterogeneity for p53 mutations in the pre-invasive area in 3 esophageal cancers. In all areas sampled in the invasive portion of each cancer, the p53 mutational status was identical in a given tumor. In heterogeneous tumors, the invasive area showed one of the p53 mutations found in the pre-invasive area. In nodal metastases, the p53 mutation was identical to that in the invasive area of each primary tumor. These data suggest that the timing of p53 alteration is not as early as might have been expected, indicating that, in regard to p53 gene alteration, some esophageal cancers are composed of various subclones in the pre-invasive stage with invasiveness developing in one of them, which becomes predominant through clonal selection.