Overexpression of HER-2/neu proto-oncogene is found in many human cancers including 20-30% of breast cancer and is a predictor of poor prognosis. To target breast cancer cells that overexpress HER-2/neu mRNA, we previously described a novel strategy that combines the principle of antisense (AS) and translational inhibitory activity conferred by an iron-responsive element (IRE) (AS-IRE). Here, we showed that three potential AS-IREs, i.e. AS-IRE1, 4, and 5, derived from HER-2/neu antisense sequence could bind endogenous iron regulatory protein (IRP) and, when placed in 5' untranslated region (5'UTR) of a reporter gene, the gene expression could be translationally repressed by recombinant IRP in vitro. Using AS-IRE4 as our model, we demonstrated that it is regulated by iron, and importantly, such regulation is impaired in HER-2/neu-overexpressing breast cancer cells. Furthermore, we showed that AS-IRE4 could preferentially direct the expression of a reporter gene in HER-2/neu-overexpressing breast cancer cells. Interestingly, when AS-IRE4 was placed in 5'UTR of Bax gene, a pro-apoptotic protein in the Bcl-2 protein family, we observed a preferential cell killing in breast cancer cells that overexpress HER-2/neu. Taken together, our results suggest that AS-IRE behaves as a functional IRE and it may direct therapeutic gene expression to preferentially target HER-2/neu-overexpressing breast cancer cells.