We previously demonstrated that delivery of a gene encoding an anti-erbB-2 intracellular single-chain antibody (sFv) resulted in down-regulation of cell surface erbB-2 levels and induction of apoptosis in erbB-2 overexpressing ovarian cancer cells. Based upon these findings, we hypothesized that human breast carcinomas overexpressing erbB-2 would be similarly affected by this genetic intervention. We evaluated the phenotypic effects resulting from intracellular expression of the anti-erbB-2 sFv on the human breast cancer cell lines MDA-MB-361, SK-BR-3, BT-474, MCF-7 and MDA-MB-231. Recombinant adeno-viruses encoding either a reporter gene (AdCMVLacZ) or the endoplasmic reticulum (ER) directed anti-erbB-2 sFv (Ad21) were delivered to various breast cancer cell lines. Cell viability was determined by a proliferation assay and fluorescent microscopy allowed visualization of apoptotic cells. An erbB-2 ELISA quantified the endogenous erbB-2 levels of each cell line. The anti-erbB-2 sFv-encoding-adenovirus, Ad21, but not the β-galactosidase encoding adenovirus, AdCMVLacZ, was cytotoxic to > 95% of the tumor cells in the MDA-MB-361 and SK-BR-3 lines, and > 60% of the tumor cells in the BT-474 line. In marked contrast, the MCF-7 and MDA-MB-231 cell lines showed no change in the rate of cell proliferation following this treatment. The cytotoxic effects generated in the first three lines were a consequence of the induction of apoptosis by the anti-erbB-2 sFv. An ELISA specific for erbB-2 showed that the breast cancer cell lines most susceptible to the anti-erbB-2 sFv, MDA-MB-361, SK-BR-3 and BT-474, overexpressed the erbB-2 protein while the cell lines demonstrating no response to the anti-erbB-2 sFv, MCF-7 and MDA-MB-231, expressed the lowest levels of erbB-2. These results demonstrate that targeted killing of erbB-2 overexpressing cells via intracellular knockout can be accomplished in the context of breast carcinoma. Furthermore, erbB-2 levels in breast tumor cells may be predictive of their sensitivity to sFv-mediated killing. The ability to accomplish selective cytotoxicity of breast cancer cell lines overexpressing the erbB-2 tumor marker should allow for derivation of clinical gene therapy strategies for breast cancer utilizing this approach.