A single blind, randomized, placebo-controlled, single-center phase I clinical trial of a CD8⁺ T-cell peptide epitope vaccine against infectious mononucleosis was conducted with 14 HLA B*0801-positive, Epstein-Barr virus (EBV)-seronegative adults. The vaccine comprised the HLA B*0801-restricted peptide epitope FLRGRAYGL and tetanus toxoid formulated in a water-in-oil adjuvant, Montanide ISA 720. FLRGRAYGL-specific responses were detected in 8/9 peptide-vaccine recipients and 0/4 placebo vaccine recipients by gamma interferon enzyme-linked immunospot assay and/or limiting-dilution analysis. The same T-cell receptor Vβ CDR3 sequence that is found in FLRGRAYGL-specific T cells from most EBV-seropositive individuals could also be detected in the peripheral blood of vaccine recipients. The vaccine was well tolerated, with the main side effect being mild to moderate injection site reactions. After a 2- to 12-year follow-up, 1/2 placebo vaccinees who acquired EBV developed infectious mononucleosis, whereas 4/4 vaccinees who acquired EBV after completing peptide vaccination seroconverted asymptomatically. Single-epitope vaccination did not predispose individuals to disease, nor did it significantly influence development of a normal repertoire of EBV-specific CD8⁺ T-cell responses following seroconversion.