Cromer blood group antigens reside on the complement regulatory protein decay accelerating factor (DAF, CD55). This glycosyl- phosphatidylinositol-anchored glycoprotein is widely distributed, especially among cell types in contact with plasma. Numerous Cromer blood group antigens have been defined using alloantibodies induced by transfusion or pregnancy. However, few pairs of antithetical antigens have been described in this system, presumably because of the rarity of the low-frequency alleles. Analysis of polymerase chain reaction- amplified genomic DNA showed that the Cr(a-) phenotype has a Ala193-- >Pro substitution in short consensus repeat 4 (SCR4) of DAF, and the Tc(a-b+) phenotype has a Arg18-->Leu substitution in SCR1 of DAF. The locations of Cra and Tca epitopes were confirmed by analysis of Chinese hamster ovary cell transfectants expressing a Cr(a-) allele-specific transfectant and a chimeric protein containing only SCR1 of DAF, respectively. Overall, these studies further show the usefulness of an approach based on recombinant proteins in mapping blood group antigen epitopes and identifying blood group antibodies.