SGN-40 is a humanized IgG1 antihuman CD40 that is currently in a phase I clinical trial for the treatment of multiple myeloma. As surface CD40 expression on B-lineage cells is maintained from pro-B cells to plasma cells, SGN-40 may be applicable to treatment of other B-cell neoplasias, including non-Hodgkin's lymphoma. In this study, we examined potential in vitro and in vivo anti-B-lineage lymphoma activity of SGN-40. Recombinant SGN-40 was expressed and purified from Chinese hamster ovary cells and characterized based on binding affinity, specificity, and normal B-cell stimulation. The ability of SGN-40 to target neoplastic B cells was examined in vitro by proliferation inhibition, cytotoxicity, and antibody-dependent cell cytotoxicity assays and in vivo by human lymphoma xenograft models. Recombinant SGN-40 showed high affinity, Kd of approximately 1 nmol/L, and specific binding to CD40. Whereas SGN-40 was a weak agonist in stimulating normal B-cell proliferation in the absence of IL-4 and CD40L, it delivered potent proliferation inhibitory and apoptotic signals to, and mediated antibody-dependent cytotoxicity against, a panel of high-grade B-lymphoma lines. These in vitro antilymphoma effects were extended to disseminated and s.c. xenograft CD40 tumor models. In these xenograft models, the antitumor activity of SGN-40 was comparable with that of rituximab. The preclinical in vitro and in vivo antilymphoma activity of SGN-40 observed in this study provides a rationale for the clinical testing of SGN-40 in the treatment of CD40+ B-lineage lymphomas.