Breast cancer cells that overexpress HER-2/neu are more resistant to chemotherapeutic agents such as paclitaxel (Taxol) and docetaxel (Taxotere) than those that do not overexpress HER-2/neu. In previous work, we showed that the adenovirus type 5 E1A can repress HER-2/neu expression at the transcriptional level. Here we first demonstrated that paclitaxel sensitivity correlates with HER-2/neu expression level in a panel of mouse fibroblasts expressing different levels of HER-2/neu, and that downregulation of HER-2/neu expression by E1A sensitizes the cells to paclitaxel. To further test whether E1A can sensitize HER-2/neu-overexpressing human breast cancer cells to paclitaxel through E1A-mediated HER-2/neu repression, an adenoviral vector was used to transfer the E1A gene into two human breast cancer cell lines, MDA-MB-453 and MDA-MB-361, that overexpress HER-2/neu. After E1A delivery, we observed that HER-2/neu expression level was reduced, and cells were treated with paclitaxel. Cell proliferation assays showed a synergistic growth inhibition effect of E1A and paclitaxel. The synergistic effect was also confirmed by soft agar colony-formation assay. Breast cancer cell lines that express low levels of HER-2/neu, MDA-MB-435 and MDA-MB-231 cells showed no synergistic growth inhibition effect when treated on the same protocols. Thus, we concluded that the adenovirus type 5 E1A gene can sensitize paclitaxel-resistant HER-2/neu-overexpressing breast cancer cells to the drug by repressing HER-2/neu expression. This in turn may have important implications for the development of a novel therapy that combines chemotherapy and gene therapy.