A continuous line of human mammary tumor cells, called 21MT, has been established in culture from a pleural effusion of a 36-year-old woman with metastatic breast cancer. The cells are epithelial as shown by morphology and expression of keratins and are mammary tumor cells as shown by expression of the HMFG-2 antigenic determinant. The cells grow well both in DFCI-1, a partially defined medium containing pituitary extract and 1% fetal bovine serum, and in alpha-minimum essential medium (alpha-MEM) supplemented with 10% serum, epidermal growth factor (EGF), insulin, and hydrocortisone. Karyotypic analysis of cells at early passage has shown the presence of rearranged (marker) chromosomes as well as aneuploidy with a net DNA content in the tetraploid range, confirmed by DNA cytofluorography, as well as double minute chromosomes in about 5% of the cells. Southern blots have revealed a 40-fold amplification of the ERBB2 gene and a 50-fold overexpression of its mRNA. The amplification of ERBB2 DNA was localized by in situ hybridization to one of the marker chromosomes but not to the double minutes. It is inferred, therefore, that at least two genes have been amplified in these cells.