The use of monoclonal antibodies (mabs) to blood group antigens is constantly increasing for routine typing. Two heterohybridoma cell lines, HMR15 and HMR22, were established by Epstein-Barr virus transformation of peripheral blood lymphocytes from a blood donor with anti-Dia. HMR15 mab directly agglutinated Di(a+) red cells, and HMR22 mab agglutinated Di(a+) red cells exclusively by the indirect antiglobulin test. Reactivities of both HMR15 and HMR22 mabs were specific for Dia and had good correlation with the reactivity of a commercial, polyclonal antiserum. The binding of monoclonal antibodies to antigen-positive red cells was mutually blocked by each other as well as by polyclonal anti-Dia. Immunoprecipitates by HMR22 mab with a Di(a+) preparation showed a 120kDa band that was stained by anti-band 3. Dia typing of 2427 blood donors with the mabs and polyclonal typing serum detected 244 Di(a+) individuals (10.1%). No discrepancy was observed between the mabs and the polyclonal antiserum. HMR15 and HMR22 mabs are useful Dia typing reagents and can substitute for commercial antiserum.