Three cytotoxic monoclonal antibodies, HU-11, HU-32, and HU-33, specific for human Ia-like antigens were used to analyze the two HLA-DR2associated HLA-D specificities, HLA-Dw2 and HLA-Dwl2. In the HLA-Dw2, DR2, MB1 homozygous B-cell line EB-CMG, the binding of radiolabeled HU-32 and HU-33 was strongly inhibited by the addition of nonlabeled HU-11, whereas no inhibition occurred in the HLA-Dw12, DR2, MB1 homozygous Bcell line EB-KT. To confirm this differential inhibition pattern further, F(ab~2 fragments were prepared from HU-11, and their ability to inhibit complementdependent lysis mediated by HU-32 and HU-33 was assessed against a total of five homozygous typing cell lines homozygous for HLA-Dw2, DR2, MBI or HLA-Dw12, DR2, MB1, including EB-CMG and EB-KT. Here again, the same d~ferential inhibition pattern as that observed in the radiobinding inhibition assays was obtained. Thus, the data suggest that the two kinds of HLA-DR2positive homozygous typing cell lines with distinct HLA-D specificity can be distinguished from each other by using solely serologic methods. This is the first clear-cut serologic distinction made between homozygous typing cells defining HLA-Dw2 and those defining HLA-Dwl2, since no serologic means that enables one to distinguish one from the other has been available.