CTL clones were derived from HLA-A2.1 transgenic mice by immunization with a human cell expressing HLA-A2.1. None of these clones lysed murine transfectants, and only 3 of 23 lysed monkey transfectants expressing HLA-A2. In contrast, all of these clones lysed a wide variety of human cells expressing HLA-A2.1. These results demonstrate the existence of species-specific epitopes on the HLA-A2.1 molecule, and suggest that these epitopes are formed by the association of class I MHC products with one or more endogenous species-specific molecules. These results provide an explanation for the frequently observed failure of HLA class I-specific CTL to recognize these antigens on murine transfectants. These results also suggest that such endogenous proteins may also contribute to the formation of epitopes recognized by allospecific CTL.