In the normal stratified squamous epithelium of the esophagus, only the third to the fifth layers of cells express the cyclin-dependent kinase inhibitor p21WAF1/CIP1 (p21). Using immunohistochemical staining, we examined the topological distribution of cells expressing p21, p53, Ki67, and cytokeratin 10 (CK10), a differentiation marker of esophageal squamous cell carcinoma (SCC), in 25 superficial SCCs and 72 dysplastic lesions of the esophagus. Image analysis of p21, p53, and Ki67 expression was also performed in 48 dysplastic lesions. In superficial SCCs, although Ki67- and p53-expressing cells were mainly distributed in the deep layers of tumors despite tumor differentiation, the distribution of p21 correlated with tumor differentiation. In dysplastic lesions, p53- and Ki67-coexpressing cells tended to locate in the same layers and expand in the lower layers of epithelium with the progression of dysplasia. p21-expressing cells shifted to the upper layers of the epithelium with the progression of dysplasia. However, this change was heterogeneous; in some lesions, p21-expressing cells were confined to the superficial layers of atypical cells (confined type), whereas in others, p21-overexpressing cells were scattered among atypical cells (scattered type). CK10 expression was observed in 25% of dysplastic lesions, and the frequency of CK10 expression was significantly higher in the scattered than in the confined type. Our results suggest that esophageal squamous dysplasia represents the earliest pathological process in esophageal squamous carcinogenesis. Our results also suggest that differentiation of esophageal SCC is determined at the stage of dysplasia, and that p21 plays a critical role in the differentiation process.