HSV-1-specific and HSV-1/HSV-2-cross-reactive H-2Kb-restricted cytotoxic T lymphocyte (CTL) clones were derived from a pool of splenic memory CTL (CTLm) obtained from HSV-1-infected C57BL/6 mice. Two of the HSV-1/HSV-2-cross-reactive CTL clones recognized HSV gB since H-2b cells infected with a recombinant adenovirus vector expressing HSV glycoprotein B (gB) provided a target for these CTL clones. The CTL recognition epitope was precisely defined as HSV-1 gB residues 498-505 using synthetic peptides and conforms to a predicted H-2Kb-binding motif. Immunization of C57BL/6 mice with the free synthetic peptide corresponding to this predicted minimal epitope (HSV-1 gB498-505) resulted in the generation of HSV-gB epitope-specific CD8+ CTL in the popliteal lymph nodes. The peptide-induced CTL recognize and lyse HSV-1 infected H-2b cells or cells pulsed with the synthetic peptide, gB498-505. The availability of CTL clones directed to this predicted minimal HSV CTL epitope should be helpful in understanding processing of HSV glycoprotein B and presentation of this CTL recognition epitope.