CAAF1 and CAAF2, newly identified calcium-binding proteins from bovine amniotic fluid, have been revealed to be members of the S100 protein family preferentially produced by fetal squamous epithelial cells, including epidermal keratinocytes. Having previously cloned the cDNA of human CAAF1 protein from the esophageal epithelium, we report here on the characteristic expression pattern of CAAF1 and related S100 proteins in human esophageal epithelial cells. Normal cells of the human esophageal epithelium expressed CAAF1, and also expressed the homologous novel S100 proteins including CAAF2, MRP8, and MRP14, but not S100alpha. An immunohistochemical study with specific monoclonal antibodies against CAAF1 proteins demonstrated that CAAF1 proteins were produced by the esophageal epithelial cells in the process of cell differentiation. The immature proliferating cells in the epithelium did not produce CAAF1 proteins, but the differentiated cells expressed CAAF1, which overlay the immature cells and were stratifying in the epithelium. These CAA 1-producing cells did not show any proliferating activities. Esophageal carcinoma cells did not express CAAF1, except for the keratinized cells with no proliferating activity. In addition, the forced expression of CAAF1 proteins in the carcinoma cells resulted in a marked decrease in DNA synthesis. These findings indicate that human esophageal epithelial cells express the multiple genes of S100 proteins including CAAF proteins, and that CAAF1 is closely associated with the terminal differentiation of these cells. CAAF1 expression also might play some role in cell growth.