SummaryThe one‐way primary equine mixed lymphocyte reaction (MLR) was used to generate allospecific major histocompatibilty complex (MHC) class‐I‐restricted cytotoxicityin vitro. Allospecific cytotoxicity, demonstrated against Con A stimulated peripheral blood mononuclear cells (PBMC) in a 6 h chromium‐release assay, was present from approximately 72 to 168 h of primary culture and peaked between 120 and 168 h. Optimal cytotoxicity was generated at a responder to stimulator ratio of 4:1 and at an effector to target ratio of 50:1. The cytotoxicity was blocked by an anti‐equine leucocyte antigen (ELA) class‐I and an anti‐equine CD8‐homologue monoclonal antibody. An anti‐ELA class‐11 and an anti‐equine CD4‐homologue monoclonal antibody ha