It is well accepted that minor histocompatibility antigens (mHag) can function as transplantation barriers between HLA-matched individuals. Little is known about the molecular nature and evolutionary conservation of mHag. It is only very recently that the first human mHag were identified. The HLA-A2.1-restricted mHag HA-2 and the HLA-B7-restricted mHag H-Y appeared to be peptides derived from polymorphic self proteins. Here we show that the HLA-A2.1-restricted mHag HA-1, HA-2, and the H-Y peptides are conserved between man, chimpanzees and rhesus macaques. Human cytotoxic T cell clones specific for the HLA-A2.1-restricted mHag HA-1, HA-2, and H-Y recognized HLA-A2.1 gene-transfected chimpanzee and rhesus macaque cells. High-pressure liquid chromatography fractionation of HLA-A2.1-bound peptides isolated from the HLA-A2.1-transfected chimpanzee cells revealed that the chimpanzee HA-1 and HA-2 co-eluted with the human HA-1 and HA-2. Subsequent amino acid sequencing showed that the chimpanzee HA-2 peptide is identical to the human HA-2 peptide. Our functional and biochemical results demonstrate that mHag peptides are conserved for over 35 million years.