The epidermal growth factor receptor (EGFR) gene was analyzed by in situ hybridization using a squamous cell carcinoma line NA, which has high numbers of EGF receptors and carries a 20-fold amplification of EGFR genes. NA cells are pseudotriploid (mode of chromosome number is 69) and have three copies of an apparently normal chromosome 7 together with several aberrant chromosomes. Strong hybridization signals were observed in the abnormal banding region of one of the aberrant chromosome, MH1, which has no structural homology to chromosome 7. This MH1 chromosome was lost in NA-derived variant lines that possess reduced numbers of EGF receptors. These results are in contrast to previous findings that EGFR gene amplification is associated with structural alterations of the short arm of chromosome 7 and provide new evidence in regard to the location of the amplified EGFR gene in tumor cells.