Functional interactions between CD8-dependent cytotoxic T cells and their targets require physical contact between CD8 and a non-polymorphic determinant on the α3 domain of the class I MHC molecule. We developed a cell-free assay to directly monitor this molecular interaction, specifically excluding the participation of other cellular proteins and lipids. This assay employed a soluble CD8 derivative and a plate-bound HLA-A2.1 derivative, α3 MalE , in which the α3 domain has been expressed independently of its neighboring polypetide domains on the native class I MHC molecule and β2-microglobulin (β2-m). These proteins were produced using eukaryotic and prokaryotic expression systems, respectively. Our data demonstrated specific, saturable binding between soluble CD8α (sCD8α) and α3 MalE , and the K_d of this interaction was determined to be 4.5 × 10⁻⁷ M. Monoclonal antibodies (mAb) directed against either CD8 or the α3 domain of class I MHC inhibited binding; mAb directed against other sites on class I MHC and β2-m did not. Our data suggest that the interaction between CD8α and the α3 domain of class I MHC does not require the participation of neighboring class I sequences or β2-m.