Functional properties of the B*4801 allotype were investigated using HLA class I-deficient 221 cells transfected with B*4801 cDNA. From pool sequence analysis of endogenously bound peptides, B*4801 was shown to select for nonamer peptides having glutamine or lysine at position 2 and leucine at the carboxyl-terminus. In an in vitro cell-cell binding assay, B*4801 binds CD8 alpha homodimers weakly due to the presence of a threonine residue at position 245 in the alpha 3 domain. A mutant B*4801 molecule in which alanine replaces threonine 245, binds CD8 alpha homodimers at levels comparable to those of other HLA class I allotypes. Despite the low affinity of B*4801 for CD8 alpha, alloreactive T-cells that recognize B*4801 molecules expressed by the 221 transfectant are inhibited by anti-CD8 monoclonal antibodies. Analysis of 25 B*48-expressing individuals from various populations showed threonine 245 was encoded by every B*48 allele.