A xenoantiserum to human Ia antigens has been described that is capable of blocking not only stimulation in the mixed lymphocyte reaction (MLR) but also the induction of cytotoxic T lymphocytes (CTLs). Data from immunofluorescence as well as complement-dependent cytolytic assays indicate that the anti-Ia xenoantiserum is directed against B cell surface antigens. Inhibition of complement-dependent cytolysis with column fractions of B cell antigens and autoradiography of immune precipitates electrophoresed on sodium dodecyl sulfate (SDS)-polyacrylamide gels have established that the antigen detected by the xenoantiserum has characteristics of the human two-polypeptide Ia molecular complex. Allogeneic stimulator cells pretreated with anti-Ia at very low doses were unable to stimulate in the MLR and failed to induce CTLs. Neither anti-beta 2-microglobulin nor a non-HLA-associated antilymphocyte serum in similar dose ranges inhibited MLR or cell-mediated lympholysis (CML) assays. Absorption of anti-Ia xenoantiserum with B lymphoblasts, but not T lymphoblasts, removed inhibitory activity for both MLR and CML. Untreated third-party stimulator cells cocultivated with anti-Ia-pretreated stimulator cells provided stimulation in the MLR that apparently allowed partial recovery of CML against targets from the same donor as the anti-Ia-treated stimulator cells. Elimination of the helper effect, normally provided by MLR stimulation, may be one mechanism by which anti-Ia xenoantiserum prevents induction of CTLs.