24 monoclonal rat antibodies are described that are reactive with determinants encoded by the major histocompatibility complex (MHC) of the rat. These hybridoma antibodies were derived by fusing mutant mouse myeloma cells to spleen cells from Lewis rats immunized with allogeneic Brown Norway cells. All 24 antibodies are cytotoxic for both Brown Norway target cells and target cells from the appropriate MHC congenic rats. Pattern of cytotoxicity and hemagglutination strongly suggest reactivity against class I (K or D equivalent) rat MHC determinants. Cytotoxic cross-reactivity patterns were generated for each monoclonal antibody on a panel of rat and mouse lymphoid cells and human peripheral T lymphocytes. A high degree of interspecies cross-reactivity was noted with approximately one-half of the antibodies positive on human and/or mouse target cells. 11 antibodies recognized polymorphic determinants in the mouse, and, by using target cells from MHC congenic mouse strains, it was shown that these determinants are encoded by genes within the H-2 complex. Finally, by considering the overall reactivity patterns of these monclonal antibodies on all target cells, one can show that these 24 antibodies represent a minimum of 14 antibody specificities.