The conditional expression of lethal genes in tumor cells is a promising gene therapy approach for the treatment of cancer. The identification of promoters that are preferentially active in cancer cells is the starting point for this strategy. The combination of tissue-specific and tumor-specific elements offers the possibility to artificially develop such promoters. We describe the construction and characterization of a hybrid promoter for transcriptional targeting of breast cancer. In many cases, breast cancer cells retain the expression of estrogen receptors, and most solid tumors suffer from hypoxia as a consequence of their aberrant vascularization. Estrogen response elements and hypoxia-responsive elements were combined to activate transcription in cells that present at least one of these characteristics. When a promoter containing these elements is used to control the expression of the pro-apoptotic gene harakiri, the induction of cell death can be activated by estrogens and hypoxia, and inhibited by antiestrogens such as tamoxifen. Finally, we show evidence that these properties are maintained in the context of an adenoviral vector (AdEHhrk). Therefore, infection with this virus preferentially kills estrogen receptor-positive breast cancer cells, or cells growing under hypoxic conditions. We propose the use of this promoter for transcriptional targeting of breast cancer.