Nearly half of HLA-A2-positive individuals in African populations have a subtype of HLA-A2 other than the A*0201 allele. We have isolated the common African HLA-A2 subtype genes from Epstein-Barr virus-transformed B cell lines and have established stable class I reduced transfectants expressing these alleles. We have studied the peptide binding and presentation properties of A*0201, A*0202, A*0205, A*0214, and A*6901 by a combination of approaches: assaying direct binding of labeled synthetic peptides, studying the ability of antigen-specific cytotoxic T lymphocytes to recognize peptide-pulsed cells, and sequencing peptide pools and individual ligands eluted from cells. We find that A*0201-restricted peptides can also bind to A*0202 but do not bind strongly to the other alleles in this study. We show that some cytotoxic T lymphocytes can recognize all subtypes capable of binding an antigenic peptide, whereas others are subtype specific. Sequencing of eluted peptides reveals that A*0202 has a similar peptide motif to A*0201, but that A*0205, A*0214, and A*6901 have different motifs. These data strongly support a model in which residue 9 (Phe or Tyr) of the A2/A68/A69 molecules is a critical factor in determining the specificity of the B pocket of the major histocompatibility complex and the position 2 anchor residue of associated peptides. We conclude that a single-amino acid difference in the major histocompatibility complex can be sufficient to cause a dramatic change in the nature of bound peptides, implying that individuals with closely related HLA subtypes may present very different repertoires of antigenic peptides to T cells in an immune response. It is likely to be a general phenomenon that very similar class I subtypes will behave as functionally distinct HLA allotypes.