HCA2 is a widely used monoclonal antibody, thought to be highly selective for HLA-A and -G heavy chains. We demonstrate here that it also shows affinity to HLA-B73 and HLA-E molecules on intact cells. By comparing the differences in the amino acid (AA) sequences of several HLA class I alleles that are either recognised or not recognised by HCA2, a likely epitope of HCA2 has been deduced. It extends from position 76 to position 83 of the alpha1-domain. In intact cells, the solvent-exposed AA in positions 76 (Ala, Val, or Met), 80 (Asn or Thr) and 83 (Gly) are likely to constitute the recognition region. Inhibition experiments with peptides spanning the region of the alpha1-domain from position 74 to 85 of various HLA class I heavy chains prove that HCA2 recognizes a broadly shared epitope on HLA-E, -F and -G molecules as well as selected HLA-A, -B and -C antigens.