Equine herpesvirus 1 (EHV-1) causes respiratory and neurological disease and abortion in horses. Animals with high frequencies of cytotoxic T lymphocytes (CTL) show reduced severity of respiratory disease and frequency of abortion, probably by CTL-mediated control of cell-associated viraemia. This study aimed to identify CTL epitopes restricted by selected major histocompatibility complex (MHC) class I alleles expressed in the equine leukocyte antigen (ELA) A3 haplotype. Effector CTL were induced from EHV-1-primed ponies and thoroughbreds with characterized MHC class I haplotypes and screened against P815 target cells transfected with selected EHV-1 genes and MHC class I genes. Targets that expressed EHV-1 gene 64 and the MHC B2 gene were lysed by effector CTL in a genetically restricted manner. There was no T-cell recognition of targets expressing either the MHC B2 gene and EHV-1 genes 2, 12, 14, 16, 35, 63 or 69, or the MHC C1 gene and EHV-1 genes 12, 14, 16 or 64. A vaccinia virus vector encoding gene 64 (NYVAC-64) was also investigated. Using lymphocytes from ELA-A3 horses, the recombinant NYVAC-64 virus induced effector CTL that lysed EHV-1-infected target cells; the recombinant virus also supplied a functional peptide that was expressed by target cells and recognized in an MHC-restricted fashion by CTL induced with EHV-1. This construct may therefore be used to determine the antigenicity of EHV-1 gene 64 for other MHC haplotypes. These techniques are broadly applicable to the identification of additional CTL target proteins and their presenting MHC alleles, not only for EHV-1, but for other equine viruses.