Recent descriptions of epitopes within HLA class I antigens recognized by mouse monoclonal antibodies are providing an antigenic map of such molecules. However, for transplantation purposes, it is crucial to understand the epitope specificity of alloantibodies. To investigate this issue, we performed sequential absorption/elution studies with serum from a broadly sensitized patient and homozygous typing cells (HTCs) which shared one HLA-A,B antigen with the patient. Antibody reactivity in the different eluates was measured by flow cytometry in a panel of 20 HTCs. These studies revealed two major findings: (a) There were multiple antibodies recognizing one HLA antigen. For example, there were 8 anti-B62 antibodies, 8 anti-B51, 5 anti-B57, 5 anti-B46, and 4 anti-B35. (b) The reactivity of most antibodies correlated highly with the presence of specific amino acids at a given position in the target HLA molecule. Such residues were absent in most HLA antigens not recognized by the antibody. Most of the target residues were located in the accessible alpha helices or connecting loops, but at least one antibody reactivity appeared to be influenced by residues located in the beta sheets. The HLA antigens evaluated in this study were those of the B5, B15, B17 cross-reactive group which have multiple epitopic sites. However, further studies are necessary to determine if alloantibodies directed to other HLA molecules will have a similar or more restricted degree of complexity.