To investigate the xenoantigenicity of HLA class I antigens, a complete series of human/mouse chimeric major histocompatibility complex class I genes was constructed by using the human HLA-B7 and mouse H-2Kbgenes. All of these hybrid antigens were stably expressed on L cells at high levels. Nine anti-HLA class I monoclonal antibodies, including 5 monomorphic, 3 broad polymorphic, and 2 specific polymorphic ones, were tested by binding to these cell lines. Our data suggest that the α2domain of HLA-B7 plays an important role in the formation of the antigenic determinants recognized by all of the anti-HLA class I mAbs tested. However, the antigenic determinants of the monomorphic mAbs were complex. In some cases, all of the three extracellular domains were required for expression of the epitope. The location of xenoimmunogenic site of HLA-B7 antigen was also examined by immunizing (B6×C3H)F1and C3H mice with the parental and hybrid antigens. Our data suggest that the a, and α2domains of HLA-B7 must be expressed together for production of antibodies against monomorphic determinants of HLA class I antigens, while the α3alone can induce xenoantibodies in (B6×C3H)F1mice but not in C3H mice.