MUC1, a heavily glycosylated mucin, has generated considerable interest as a target for tumor killing because of its overexpression in malignancies. Full-length MUC1 (MUC1/TM) is proteolytically cleaved after synthesis generating alpha and beta subunits, which specifically bind in a noncovalent interaction. Although the beta chain remains on the cell surface, the alpha chain binds in an on-and-off interaction. Most anti-MUC1 antibodies (Abs) described to date recognize epitopes within the highly immunogenic alpha-chain tandem repeat. Because the alpha-chain is shed, such Abs are sequestered and fail to reach MUC1-expressing cells. Immunizing with cDNA encoding MUC1/TM and the spliced MUC1/X isoform from which the tandem repeat has been deleted yielded antibodies to the MUC1 alpha/beta junction. Pseudomonas toxin PE38 linked to polyclonal anti-MUC1 alpha/beta junction Abs both bound and killed MUC1-positive malignant cells. Monoclonal DMC209 binds the MUC1 alpha/beta junction in both MUC1/X and MUC1/TM. When injected into SCID mice xenotransplanted with human breast cancer MDA-MB-231, monoclonal DMC209 showed significant in vivo tumor-suppressive activity. The MUC1/X alpha/beta junction presents a biologically-significant target in MUC1-expressing malignancies because (i) antibodies directed against cell-bound alpha/beta junction epitopes reach the intended cellular target, (ii) antibodies to junction epitope are internalized into cells, (iii) anti alpha/beta junction antibodies can effectively kill high MUC1-expressing cancer cells as antibody-toxin conjugates and (iv) antibodies targeting the MUC1 cell-bound alpha/beta junction results in tumor suppression in vivo. Our results indicate that cell-bound MUC1 alpha/beta junction, unlike shed alpha chain, represents a highly effective moiety for targeting and killing MUC1-expressing malignancies.