The Dombrock (Do) glycoprotein is a glycosylphosphatidylinositol(GPI)-linked membrane protein carrying Dombrock blood group antigens. There are no standardized typing reagents for Do(a) or Do(b). We have developed ten different monoclonal antibodies(MoAbs) that are specific for Dombrock. The objectives of this study were to characterize these MoAbs serologically and determine the epitopes they recognize. MoAbs were generated by standard fusion methods. Mice were immunized with transfected human embryonic kidney 293T cells expressing high levels Do(a) or Do(b). The MoAbs were tested serologically with untreated and enzymatically or chemically modified red blood cells (RBCs).Serologic inhibition studies were performed with synthetic peptides corresponding to Do(a) and Do(b) amino acid sequences.Pepscan epitope analysis was done on an array of immobilized tridecapeptides corresponding to the full-length polypeptide. All ten antibodies were serologically specific for Dombrock. Eight of the antibodies recognized epitopes that were resistant to treatment with ficin, pronase, a-chymotrypsin, and neuraminidase,but sensitive to trypsin and 0.2 M dithiothreitol (DTT). Five have anti-Do(b)-like specificity. The epitope recognized by MIMA-52 was neuraminidase sensitive, and MIMA-127 epitope recognized a DTT-resistant, linear epitope (90)QKNYFRMWQK(99) of the Dombrock polypeptide. MIMA-127 was the only one of the ten Dombrock MoAbs mapped to a specific sequence of the Dombrock glycoprotein; the other nine MoAbs did not provide aspecific peptide binding pattern. The other MoAbs could not be mapped as they most likely recognize nonlinear, conformation-dependent epitopes, as is evident by their sensitivity to reduction of disulfide bonds by DTT. The dependence of some epitopes on antigen glycosylation is also a possibility.