To develop an effective tumor immunotherapy for B-lineage non-Hodgkin's lymphoma (NHL) and acute lymphoblastic leukemia (ALL), a bispecific monoclonal antibody (BsAb) has been generated with the first specificity for the CD3 epsilon-chain and the second for the CD19 antigen. Peripheral blood mononuclear cells (PBMCs) isolated from patients with NHL or ALL during remission or relapse rapidly proliferated (up to 179-fold increase) on in vitro activation combining phytohemagglutinin or CD3 monoclonal antibody with interleukin-2. After 3 weeks of stimulation, more than 90% of the PBMCs was CD3+ and CD8+, even when cultures were started with only 5% CD3+ cells. Cytotoxic activity against autologous malignant B cells was markedly enhanced (from 5% baseline to 70% lysis) by the addition of the CD3 x CD19 BsAb in all samples tested. Immunophenotypic examination of a series of tumor target cells showed that all samples examined showed CD54 (intercellular adhesion molecule-1) and HLA class I, but showed no B7 expression. CD11a (lymphocyte function-associated antigen-1) expression was heterogeneous. Various types of experiments showed that efficient CD3 x CD19 BsAb-mediated cytolytic capacity was not dependent on expression of either of these surface proteins. This contrasts with normal major histocompatibility complex-restricted antigen-specific cytotoxicity and may be essential for effective in vivo application of this BsAb.