Mechanisms that result in HER2/neu over-expression in breast cancer were examined by studying breast cancer cell lines that express much higher levels of HER2/neu mRNA than normal breast tissue while maintaining a near normal HER2/neu gene copy number. Nuclear run-on experiments indicate that the breast cancer cell lines MDA-MB453, BT483 and BT474 have an increased HER2/neu gene transcription rate. By using HER2/neu promoter-CAT constructs, we show that the enhanced HER2/neu transcription rate in MDA-MB453 is due to activation of the gene in trans. We have localized a 13-bp element on the gene promoter that is required for the increased transcription rate and have demonstrated sequence specific interaction of this fragment with a nuclear protein complex. We have also shown that in BT474 cells, transcriptional upregulation is mainly due to gene amplification and does not fully account for the levels of HER2/neu mRNA, demonstrating that deregulation of HER2/neu expression at the post-transcriptional level significantly contributes to HER2/neu overexpression in BT474 cells. Our results suggest that multiple activations of the HER2/neu gene are involved in HER2/neu over-expression in breast cancer lines and that multiple mechanisms may function simultaneously within a single cell line.