Techniques are reported for the induction and assay of cytotoxic effector cells capable of specifically lysing hapten-coupled EL4 leukemia targets. It is shown that EL4 cells survive coupling with TNP-sulfonic acid and retain the hapten on their cell surface for a prolonged period of time. Although TNP-EL4 cells are readily lysed by anti-TNP serum in a complement-mediated reaction, they are inefficiently killed in an antibody-dependent cell-mediated reaction. Cytotoxic effector cells, able to lyse TNP-EL4 targets, are induced when C57BL/6 spleen H-2-b cells are cultured with the following cell types which have been coupled with TNP: 1) ALLOGENEIC P815 tumor cells (H-2-d), 2) syngeneic EL4 tumor cells, 3) allogeneic BALB/c spleen cells (H-2-d), 4) syngeneic C57BL/6 spleen cells. Further experiments show that TNP-coupled xenogeneic chicken erythrocytes, which by themselves are unable to induce cytotoxic effectors, are capable of doing so if uncoupled P815 cells are present simultaneously. On the basis of these findings, it can be hypothesized that two stimuli are required for induction of these cytotoxic effector cells--one provided by the hapten, and the other by the P815 cell. Treatment of cytotoxic spleen cells induced by hapten-coupled allogeneic tumor cells with anti-Thy-1 serum and complement abrogates their cytotoxicity, indicating that T cells play a central role in the cytotoxic reaction. TNP-coupled erythrocytes do not serve as targets for these cytotoxic T cells, but do cause competitive inhibition of TNP-EL4 when added to the reaction mixture at high ratios. However, because the inhibition is relatively low, and because no such inhibition can be demonstrated with TNP-lysine, it is concluded that the receptor on the cytotoxic effector cell has a low affinity for hapten. This low affinity could be due to the receptor recognizing an antigen comprising mouse cell surface antigen in addition to the TNP moiety. Supporting this interpretation is the finding that TNP-EL4 cells competitively inhibit cytotoxicity much more efficiently than TNP-CRBC and that even uncoupled EL4 cells inhibit to some extent.