Cytotoxic T-lymphocyte (CTL) responses to Epstein-Barr virus (EBV) tend to focus on a few immunodominant viral epitopes; where these epitope sequences are polymorphic between EBV strains, host CTL specificities should reflect the identity of the resident strain. In studying responses in HLA-B27-positive virus carriers, we identified 2 of 15 individuals who had strong CTL memory to the pan-B27 epitope RRIYDLIEL (RRIY) from nuclear antigen EBNA3C but whose endogenous EBV strain, isolated in vitro, encoded a variant sequence RKIYDLIEL (RKIY) which did not form stable complexes with B27 molecules and which was poorly recognized by RRIY-specific CTLs. To check if such individuals were also carrying an epitope-positive strain (either related to or distinct from the in vitro isolate), we screened DNA from freshly isolated peripheral blood mononuclear cells for amplifiable virus sequences across the EBNA3C epitope, across a different region of EBNA3C with type 1-type 2 sequence divergence, and across a polymorphic region of EBNA1. This showed that one of the unexplained RRIY responders carried two distinct type 1 strains, one with an RKIY and one with an RRIY epitope sequence. The other responder carried an RKIY-positive type 1 strain and a type 2 virus whose epitope sequence of RRIFDLIEL was antigenically cross-reactive with RRIY. Of 15 EBV-seropositive donors analyzed by such assays, 12 appeared to be carrying a single virus strain, one was coinfected with distinct type 1 strains, and two were carrying both type 1 and type 2 viruses. This implies that a small but significant percentage of healthy virus carriers harbor multiple, perhaps sequentially acquired, EBV strains.