Promoter elements accounting for HER2 (c-erbB-2/neu) overexpression were searched for in several human breast cancer cell lines (MDA-453, BT-474, ZR-75-1, MCF-7) known to express constitutively a 30-fold range in HER2 transcripts per gene copy. HER2 overexpressing cells showed a single prominent DNase I hypersensitive site near a conserved and hitherto unrecognized ets response element (GAGGAA), located 38 bases down-stream from the CAAT box and directly 5' of the TATA box in the human HER2 promoter. Transient transfection of HER2 promoter constructs (0.125, 0.5, and 2.0 kilobase pairs (kb)) demonstrated that the most proximal promoter region (0.125 kb) was capable of conferring up to 30-fold enhanced activity in HER2-overexpressing cell lines relative to low HER2-expressing control lines. Site-directed mutagenesis of the ets response element (GAGGAA-->GAGAGA) caused a > or = 60% reduction in promoter activity affecting at least 0.5 kb of upstream HER2 regulatory sequence. Gel-shift assays with nuclear extracts and oligonucleotide sequences spanning the 0.125-kb promoter region detected an ETS-immunoreactive complex, present most abundantly in cells overexpressing HER2, whose high-affinity binding depended on the GAGGAA response element. Methylation interference confirmed the ETS-specific pattern of protein binding by this complex to guanine bases in the ets response element. UV cross-linking and immunoprecipitation implicate a approximately 60-kDa ETS protein, and candidate ETS genes expressed in these breast cancer cells include GABP alpha, elk-1, elf-1, and PEA3.