Background: Targeted immunotherapy, based on antibodies against tumor-associated antigens, is a promising approach for the treatment of multiple myeloma (MM). Recently, antibody-based strategies delivering a toxic payload have documented impressive clinical activity in hematological malignancies. In particular, surface molecules overexpressed on malignant plasma cells and efficiently internalized represent promising targets for developing myeloma-directed immunoconstructs. Here, the identification of CD317 (HM1.24) as a potent target structure and the characterization of a novel CD317-directed single-chain immunotoxin, HM1.24-ETA', is described. Methods: Using a novel screening tool, a panel of antibodies against MM-associated antigens was evaluated for their ability to mediate antigen-dependent delivery of a truncated version of Pseudomonas exotoxin A (ETA’) to MM cells. HM1.24-ETA' was generated by genetic fusion of a CD317-specific single-chain Fv antibody and ETA'. The anti-myeloma activity of the E. coli-expressed immunotoxin was evaluated in vitro and in a xenograft mouse model. Results: By screening a panel of antibodies including CD38, CS1, IL-6R, CD138 and CD317, CD317 was identified as a suitable receptor to deliver ETA’ to MM cells. The subsequently designed recombinant HM1.24-ETA' immunotoxin efficiently inhibited growth of MM cell lines with halfmaximal growth inhibition at concentrations of less than 1 nM. Antigen-specific MM cell killing occurred via induction of apoptosis. The proliferation of IL-6 dependent INA-6 cells was completely inhibited by HM1.24-ETA' even in the presence of bone marrow stromal cells that otherwise strongly support tumor cell growth. Importantly, HM1.24-ETA' strongly triggered apoptosis (up to 80%) in freshly isolated tumor cells from 7 out of 7 MM patients. In a xenograft SCID mouse model, establishment of INA-6 plasma cell tumors was efficiently abrogated by treatment with HM1.24-ETA' immunotoxin (p < 0.04). Conclusions: The HM1.24-ETA' immunotoxin in vitro and in the preclinical xenograft model in vivo demonstrates that the CD317 antigen may represent a promising target structure for immunotherapy of MM using immunoconjugates with toxic payloads.