The cell surface molecules CD4 and CD8 greatly enhance the sensitivity of T-cell antigen recognition, acting as "co-receptors" by binding to the same major histocompatibility complex (MHC) molecules as the T-cell receptor (TCR). Here we use surface plasmon resonance to study the binding of CD8 alpha alpha to class I MHC molecules. CD8 alpha alpha bound the classical MHC molecules HLA- A*0201, -A*1101, -B*3501, and -C*0702 with dissociation constants (K sub(d)) of 90-220 mu M, a range of affinities distinctly lower than that of TCR/peptide-MHC interaction. We suggest such affinities apply to most CD8 alpha alpha /classical class I MHC interactions and may be optimal for T-cell recognition. In contrast, CD8 alpha alpha bound both HLA-A*6801 and B*4801 with a significantly lower affinity ( greater than or equal to 1 mM), consistent with the finding that interactions with these alleles are unable to mediate cell-cell adhesion. Interestingly, CD8 alpha alpha bound normally to the nonclassical MHC molecule HLA-G (K sub(d) similar to 150 mu M), but only weakly to the natural killer cell receptor ligand HLA-E (K sub(d) greater than or equal to 1 mM). Site-directed mutagenesis experiments revealed that variation in CD8 alpha alpha binding affinity can be explained by amino acid differences within the alpha 3 domain. Taken together with crystallographic studies, these results indicate that subtle conformational changes in the solvent exposed alpha 3 domain loop (residues 223-229) can account for the differential ability of both classical and nonclassical class I MHC molecules to bind CD8.