Gene amplification/overexpression was analyzed in 23 cell lines derived from human esophageal squamous-cell-carcinoma tissues by Southern and Northern hybridizations to c-myc, c-erbB, hst-1 and cyclin-D1 probes. Amplification of the c-myc gene was observed in 5 cell lines derived from well-differentiated carcinomas and all of them were accompanied by co-amplification of other examined oncogenes. The c-erbB gene was amplified in 3 cell lines. Co-amplification of hst-1 and cyclin D1, both of which are located in chromosome 11q13, was found in 9 cell lines. Without exception their amplification was simultaneous and the magnitudes were similar. Their amplification, but not their overexpression, was significantly correlated with poor prognosis in patients from whom the cell lines were established. While hst-1-gene expression was not detected, at least 1 of the genes analyzed was overexpressed in 20 cell lines vs. its expression in normal esophageal mucosal tissues. However, gene amplification was not necessarily accompanied by overexpression of the corresponding genes. Expression of the cyclin D1 gene, which has been assumed to be a target gene for 11q13 amplification, was not detected in one particular cell line with amplification of 11q13. These results suggest that the amplification/overexpression of more than I oncogene is involved in the carcinogenic process of esophageal carcinoma and that c-myc-gene amplification is associated with a well-differentiated subtype. There remains a possibility that key oncogenes other than cyclin D1 are involved in 11q13 amplification.