Equine herpesvirus-1 (EHV-1) causes serious disease in horses throughout the world, despite the frequent use of vaccines. CTLs are thought to be critical for protection from primary and reactivating latent EHV-1 infections. However, the antigen-specificity of EHV-1-specific CTLs is unknown. The aim of this study was to identify EHV-1 genes that encode proteins containing CTL epitopes and to determine their MHC I (or ELA-A in the horse) restriction. Equine dendritic cells, transfected with a series of EHV-1 genes, were used to stimulate autologous CTL precursor populations derived from previously infected horses. Cytotoxicity was subsequently measured against EHV-1-infected PWM lymphoblast targets. Dendritic cells were infected with EHV-1 (positive control) or transfected with plasmids encoding the gB, gC, gD, gE, gH, gI, gL, immediate-early (IE) or early protein of EHV-1 using the PowderJect XR-1 research device. Dendritic cells transfected with the IE gene induced CTL responses in four of six ponies. All four of these ponies shared a common ELA-A3.1 haplotype. Dendritic cells transfected with gC, gD, gI and gL glycoproteins induced CTLs in individual ponies. The cytotoxic activity was ELA-A-restricted, as heterologous targets from ELA-A mismatched ponies were not killed and an MHC I blocking antibody reduced EHV-1-specific killing. This is the first identification of an EHV-1 protein containing ELA-A-restricted CTL epitopes. This assay can now be used to study CTL specificity for EHV-1 proteins in horses with a broad range of ELA-A haplotypes, with the goal of developing a multi-epitope EHV-1 vaccine.