A cytotoxic murine monoclonal antibody, designated P5.1, was tested against 613 unrelated donors and found to react with 401 who were positive for HLA-A2 (sensitivity = 100%) and with 8 of 82 positive for HLA-A28. The latter split of A28 corresponds to the "A28* variant" that in the Ninth International Histocompatibility Workshop (9WS) was designated Aw69(28*). The epitope recognized by antibody P5.1 is distinct from the alloantisera-defined determinants that characterize HLA-A2 and A28. Immunoprecipitation of specific antigens with selected monoclonal antibodies and isoelectric focusing gel electrophoresis demonstrated that A2, Aw68(28) and Aw69(28*) are distinct polypeptides. Thus, the A2-A28 antigen family consists of at least three different alleles definable using alloantiserums specific for A2 and A28, and monoclonal antibodies such as P5.1 recognizing the A2,Aw-69(28*)-epitope.