The HLA-A2 antigen expressed by donor OZB can be distinguished from the main HLA-A2.1 subtype by isoelectric focusing - it is one charge unit more acidic - and by some alloreactive T-cell clones but not by cytolytic T lymphocyte lines. The structure of variant OZB has been examined by comparative peptide mapping with A2.1 and radiochemical sequence analysis. The two molecules were found to differ in a single tryptic peptide from the 0 region, spanning residues 220-243. The amino acid sequence of this peptide from variant OZB revealed that there was only one amino acid change of Glu instead of Ala at position 236, a hitherto invariant residue in class I HLA antigens. All previously characterized HLA or H-2 natural variants have structural changes restricted to the α 1 and/or α2 domains. Thus, variant OZB is unique in that (1) it has one amino acid change in α3 and (2) it has no changes in αl and α2. The only detected substitution of this variant may be accounted for by a single base change at the DNA level, suggesting that it might have resulted from a point mutation in the A2.1 gene. The structural features of variant OZB open a novel way to examine the influence of polymorphism in α3 on cytolytic T-cell recognition of naturally occurring class I antigens.