The monomorphic cell surface glycoprotein CD8 acts as co-receptor in the recognition of peptide-major histocompatibility complex (MHC) class I complexes by cytotoxic lymphocytes (CTL) by binding to the monomorphic alpha 3 domain of the class I molecule. Positions 227 and 245 in the class I alpha 3 domain appear to be especially important for this interaction. Recent reports suggest there is no interspecies recognition between CD8 and MHC class I. In this study, hybrid genes from human class I HLA-A0201 and murine class I H-2Kb were transfected into human and mouse cells and tested in Cr-release assays using HLA-A0201-restricted influenza A matrix peptide-specific CTL as effectors. Transfected cells expressing chimeric genes comprising the alpha 1 and alpha 2 domains from HLA-A0201 together with the H-2Kb alpha 3 domain were lysed as effectively as wild-type HLA-A0201 and in both cases, killing was blocked by anti-CD8 antibody equally well. These results indicate that human CD8 can interact with the alpha 3 domain of murine class I to the same level as human class I.