Bi-specific monoclonal antibodies (MAbs) were developed by somatic hybridization of 2 mouse hybridomas, one producing MAb against the G250 renal-cell carcinoma (RCC)-associated antigen and the other against the T-cell antigen CD3 (OKT3). The dual specificity of the hybrid MAb produced by these so-called quadromas was analyzed by immunohistochemistry on tissue sections and by cytotoxicity assays with relevant target and effector cells. The bi-specific MAb could induce TCR alpha beta/CD3+ and TCR gamma delta/CD3+ cloned lymphocytes to kill RCC cells. A noteworthy finding was that the TCR alpha beta and gamma delta lymphocyte clones showed different triggering abilities. The specificity of target-cell lysis by the cytotoxic T cells (CTL) was dictated by the specificity of the G250 MAb. Control bi-specific MAb, recognizing a cell-surface structure not involved in T-cell activation, did not induce lysis. Several IgG subclass switch variants of the G250 hybridoma, i.e., IgG1, 2a, 2b and IgE, were used for somatic hybridization with the OKT3 hybridoma (IgG2a). Except for IgE, all IgG subclass combinations could equally induce cytolysis. Induction of cytolysis was inhibited only by excess OKT3 MAb. Comparison of 2 bi-specific MAb preparations of the same combination (IgG2a/1), produced by 2 quadromas derived from the same parental hybridomas after identical purification procedures, produced different amounts of bispecific MAb.