The PEA3 group of transcription factors belongs to the ets family and is composed of 3 known members, PEA3, ERM and ER81, which are more than 95% identical within the DNA-binding ETS domain and exhibit 50% aa identity overall. Recently, transgenic mice bearing the c-erbB-2/neu oncogene have been shown to over-express PEA3 mRNA in mammary adenocarcinomas, suggesting a role for this gene family in mammary tumorigenesis. In the present work we characterized the mRNA expression levels of PEA3-group genes in a series of human epithelial breast cell lines. Each of the 3 genes was highly expressed in normal human HMEC 1001-7 and HMEC 219-4 cells. In breast-cancer cell lines, the 3 genes were highly expressed in the ER- MDA-MB-436, MDA-MB-330, MDA-MB-231 and BT-20 cell lines, but not in the ER+ MDA-MB-134-VI and ZR-75-1 cells. In an attempt to characterize the PEA3-group proteins in breast-cancer cells, we first produced and characterized specific antibodies against each of these 3 proteins. The anti-ERM and anti-ER81 antibodies recognized specific strong bands at approximately 72 kDa and 62 kDa, corresponding to ERM and ER81, respectively, in MDA-MB-231 and Hs-578T cells expressing significant levels of the 3 mRNAs. No protein was detected in MCF-7 cells expressing low levels of mRNA for PEA3-group-family genes, or in ZR-75-1 cells, where mRNA was undetectable by Northern blot. Although in vitro-translated PEA3 is specifically immunoprecipitated by anti-PEA3 anti-serum, we were unable to immunoprecipitate PEA3 protein from MDA-MB-231 and Hs-578T cells. In order to study the transcription factor activity of ERM, PEA3 and ER81 proteins in mammary-cancer cells, we tested their ability to transactivate a reporter plasmid containing 3 Ets-binding sites, and were able to show that, in all the breast-cancer cells tested, transfected ERM, PEA3 and ER81 are able to transactivate. Although the target genes of the PEA3 group of transcription factors in breast-cancer cells have yet to be determined, these genes have a potential role in the regulation of growth and the progression of human breast cancer.