Molecular evidence has been obtained for a novel monomorphic HLA class II molecule distinct from HLA-DP/DQ/DR using a panel of lymphoblastoid cells which include HLA-loss mutants. The expression of this molecule was investigated using monomorphic affinity-purified mouse monoclonal antibodies (mAbs), including one of the IgG2a subclass designated EDU-1. This antibody reacts strongly in a cell-binding radioimmunoassay with HLA-DR and -DQ loss mutants derived from a lymphoblastoid parental cell. The EDU-1 mAb also reacted with a local panel of homozygous Epstein-Barr virus-transformed cell lines. The reactive molecules were further detected on allostimulated T-cell clones and various leukemic cells including those of myeloid origin which lack surface expression of HLA-DQ molecules. Thus the class II molecule described in this study corresponds to a monomorphic HLA class II determinant expressed on a variety of cells of different origin and HLA phenotypes. Moreover, this antigen structure is distinct from that of HLA-DP/DQ/DR as shown by direct immunoprecipitation, serial immunodepletion experiments, and two-dimensional gel electrophoresis. The molecule could be specified by new class II genes between DP and DQ. An alternative explanation for the genetic basis of the novel molecule is the existence of isotypic associations between alpha and beta chains of various class II molecules (DP, DX, DZ, and DO) but not DR and DQ as the mutant cells tested lack the latter genes.