The T cell-mediated cytotoxic response against autologous cells modified with the sulfhydryl reagent I-AED (N-iodoacetyl-N'-(5-sulfonic-1-naphthyl) ethylene diamine) is hapten specific and H-2 restricted (Levy, R. B., Shearer, G. M., Richardson, J. C. and Henkart, P. A., J. Immunol. 1981. 127: 523). We have produced a monoclonal antibody (V-6-3, IgM) which binds to AED-modified cells and proteins. Competition experiments by free hapten indicated that the binding was AED specific. The effect of the mAb on AED-specific cytotoxic T cell recognition at the effector and induction stage has been examined. Anti-AED mAb inhibited the cell-mediated lysis of some but not all AED-specific, H-2b-restricted long-term cytotoxic T cell clones and of bulk-cultured C57BL/6 anti-AED-self effector cells. This blocking was not due to nonspecific agglutination of targets since lysis of AED-modified target cells by alloreactive effector cells was not affected by this mAb under comparable conditions. Furthermore anti-AED mAb specifically inhibited the antigen-induced proliferation of AED-specific long-term cytotoxic T cell clones and the generation of AED-specific cytotoxic effector cells in secondary cultures. This monoclonal anti-AED antibody bound to cells modified by the recently described aminoreactive reagent AED-NH2 (Takai, Y., Mizuochi, H., Fujiwava, H. and Hamaoka, T., J. Immunol. 1984. 132: 57); these same target cells were, however, not lysed by AED-SH-specific cytotoxic T cell clones.