Two monoclonal antibodies (mAb 41B3 and mAb 51B3), directed against hen's egg ovomucoid (OM) and different from those we previously reported (mAb 23£5 and mAb 32A8), were prepared and purified. A competitive radioimmunoassay using 12sI_labeled OM showed that both mAb 41B3 and mAb 51B3 could bind all three homologous domains (domains I (DI), II (DII) and III (DIll)) of OM and that they reacted most efficiently with DIll. To analyze the paratope specificities of these four mAbs, a sandwich assay and a competitive radioimmunoassay were done. Only the pair mAb 41 B3 and mAb 51 B3 could not simultaneously bind the OM or domains in the sandwich assay. Only mAb 41B3 inhibited the binding of mAb 51B3 to antigens and vice versa in the competitive radioimmunoassay. These results suggest that mAb 41B3 and mAb 51B3 recognized a closely related site distinct from the epitopes for mAb 23£5 or mAb 32A8. These mAbs may be useful for general studies on epitopes of protein antigens as well as for analyses of the antigenic determinants of OM.