The monoclonal antibody GAP A3 detects the HLA allospecificity A3. Reactivity of the monoclonal was in exact concordance with the presence of the A3 antigen as defined by conventional alloantisera on a panel of 59 cells from individuals with well-characterized HLA antigens. Reactivity with GAP A3 segregated with HLA-A3 in a family where three of eight siblings inherited the paternal A3 antigen. GAP A3 precipitated appropriate 44,000- and 12,000-dalton bands on SDS-polyacrylamide gels under reducing conditions from an HLA-A3-positive, but not an HLA-A3-negative B lymphoblastoid cell line. Thus, by serological, familial, and biochemical criteria, GAP A3 defines the allospecificity HLA-A3.