The products of both CDKN2 and cyclin D1 genes are negative and positive regulators respectively in cell cycle. To study the involvement of the CDKN2 tumor suppressor gene and cyclin D1 oncogene in esophageal cancer development, we examined the situation of both genes in 21 pairs of primary human esophageal cancers and the mucosa adjacent to the cancers and also in four esophageal cancer cell lines by means of molecular biological and immunohistochemical techniques. Homozygous deletion was observed in 6 out of the 21 primary cancers and with lymph node metastasis in 5 out of the 6 cases. Loss of expression of p16 protein was identified immunohistochemically in 8 out of 21 primary cancers. Amplification of cyclin D1 was found in 12 out of 21 primary cancers and in 5 esophageal mucosa adjacent to the tumors, accompanying with overexpression of cyclin D1 protein. Homozygous deletion was observed in one (EC8712) out of the four cell lines, while by Northern and immunohisto chemistry analysis, loss of transcription of CDKN2 mRNA and loss of p16 protein expression were observed in two cell lines (EC8712 and EC8501). Amplification and overexpression of cyclin D1 were found in two cell lines (EC8733 and EC8501). These findings suggest that loss of CDKN2 gene and amplification of cyclin D1 gene are involved in esophageal cancers and that cyclin D1 alteration may be a earlier molecular event while CDKN2 to be a later one.