We have recently described a new method of eliminating graft-versus-host-disease–(GVHD)–causing T cells from murine lymphohematopoietic grafts across major histocompatibility barriers. This method was complement-independent and protected H-2-mismatched recipients long-term from the lethal effects of GVHD. The method utilized donor cell pretreatment with a cell- specific cytotoxic reagent obtained by covalently linking monoclonal anti-Thy-1 antibody to the toxin ricin. In the present study, we investigated in a more detailed manner the selectivity of anti-T-cell antibody-toxin conjugates. We found that anti-Thy-1.2-ricin conjugates were at least 30-fold more effective in killing murine T cells than pluripotential stem cells measured by colony-forming unit–spleen assay. We investigated the selectivity of both anti-Thy-1.2-ricin and anti-Thy-1.1-ricin hybrids. MLR assays showed anti-Thy-1.2-ricin was 11–100 times more toxic to Thy-1.2-positive BALB/c responder cells than Thy-1.1-positive AKR responders. In contrast, anti-Thy-1.1-ricin hybrids were more than 100-fold more toxic to Thy-1.1-positive AKR responders than to BALB/c responders. In addition to studies with anti-T-cell antibody linked to intact ricin, we also examined the activity of anti-Tcell antibody linked to ricin A chain. Anti-Thy-1.1–A chain pretreatment of AKR splenocytes resulted in inhibition of T cell mitogenesis induced by PHA. Inhibition was selective for AKR cells because the proliferation of BALB/c cells stimulated with PHA was not reduced. However, the pretreatment of AKR cells with antibody-A chain reagent resulted in 20–117 times less inhibition than pretreatment with antibody linked to intact ricin, suggesting that the antibody-A chain conjugate was much less efficient. The utility of anti-Thy-1.2-intact ricin conjugates as reagents for GVHD prophylaxis was shown in a model of fatal GVHD in which BALB/c bone marrow cells plus splenocytes were given to irradiated C57BL/6 recipients. All of these mice died by 4 months posttransplant. More than 80% of the animals were protected when their grafts were pretreated with lactose plus 500 ng/ml Thy-1.2-ricin, a dose selected on the basis of the in vitro experiments. Animals successfully engrafted and were long-term chimeras. Dual skin grafting experiments at 62 days posttransplant showed the “anti-Thy-1.2-ricin chimeras” could reject third-party grafts while tolerating donor-type grafts. In these studies, the specificity of anti-T-cell-ricin conjugates in eliminating GVHD following allogeneic bone marrow transplants was correlated with the differential toxicity towards T lymphocytes and pluripotential stem cells. Similar assays should prove valuable in designing a clinically useful antihuman T cell conjugate.