Water-soluble classical (RT1-A) class I MHC molecules were purified from aqueous extracts of DA strain liver. Following monoclonal antibody affinity, lentil lectin affinity, and gel filtration chromatography, 600 μg of soluble RT1-A class I molecules with antigen activity equivalent to 1.3×1011nucleated DA spleen cells (>500 DA spleens) was obtained. Both PVG and LEW strain recipients of DA kidney allografts were pretreated with intravenous injections of the DA soluble class I molecules, in doses with antigen activity equivalent to 108nucleated DA spleen cells. Three protocols of pretreatment were used: twice-weekly injections for 4–5 weeks, with grafting 3 or 4 days after the last injection; a single injection 7 days pregraft; or a single injection 1 day pregraft. The PVG and LEW rats received the soluble class I pretreatment either alone or in combination with suboptimal doses (2 mg/kg/day) of cyclosporine after grafting, making a total of 12 experimental groups treated with soluble class I antigen. In no case did treatment with soluble class I antigen elicit an antibody reponse in prospective graft recipients; influence kidney graft survival in any way; or enhance or suppress the antibody response to the kidney graft.The soluble DA class I MHC molecules were tested in vitro for their effect on the generation and effector function of allospecific PVG and LEW anti DA RT1-A class I cytotoxic T cells and TNP specific, self RT1-A restricted cytotoxic T cells. Concentrations up to 5 μg/ml (10−7M), equivalent to 109nucleated DA spleen cells/ml, were without any efffect.We conclude that monomeric forms of water-soluble classical class I molecules are poor immunogens—and, at doses conventionally used for active enhancement, do not influence cytotoxic T cell responses and have little potential for donor-specific immunosuppression.