The phenomenon of T cell allorecognition is difficult to accommodate within the framework of a T cell repertoire positively selected in the thymus, unless allorecognition results from the cross-reactions of self-major histocompatibility complex restricted T cells. Herein, we demonstrate the dual specificity of cytotoxic T lymphocyte (CTL) clones for the immunodominant Epstein-Barr virus (EBV) epitope FLRGRAYGL, presented on HLA-B8, and the alloantigen HLA-B*4402. CTL which recognized peptide FLRGRAYGL in association with HLA-B8 could be reactivated in vitro from healthy individuals who had been exposed previously to EBV, using stimulator cells expressing the cross-reacting alloantigen HLA-B*4402. Limiting dilution analysis of the alloresponse to HLA-B*4402 in eight healthy individuals revealed that HLA-B8+, EBV-sero+ donors had higher CTL precursor frequencies for alloantigen HLA-B*4402 than EBV-sero- control donors. It is surprising that the majority (65-100%) of anti-HLA-B*4402 CTL, generated in limiting dilution mixed lymphocyte reactions between responder cells from HLA-B8+, EBV-sero+ individuals and HLA-B*4402+ stimulators, also recognized the EBV CTL epitope FLRGRAYGL/HLA-B8. In contrast to previous studies showing extensive diversity in the T cell repertoire against individual alloantigens, these data demonstrate that the response to an alloantigen can be dominated by CTL cross-reactive with a single viral epitope, thus illustrating a possible mechanism for the frequent clinical association between herpesvirus exposure and graft-versus-host disease after bone marrow transplants.