We describe the generation and characterization of a murine monoclonal antibody with broad anti-HLA-DR beta activity, but which does not recognize DRw11/Dw5. A BALB/c mouse was immunized with a human alloreactive T-cell clone, with the intention of generating monoclonal anti-T-cell-receptor antibodies. In the course of screening the resulting hybridomas, a clone was detected which secreted an antibody (MP2) with anti-HLA-DR activity. This was shown by flow cytometry as well as immunoprecipitation followed by gel electrophoresis. Flow cytometry experiments using transfectants bearing hybrid human/murine class II molecules demonstrated that MP2 binds to the DR beta chain. MP2 bound to a wide range of Epstein-Barr-Virus-transformed cell lines and transfectants expressing different DR beta 1 and DR beta 3 subtypes: the only exceptions were three transfectants expressing DRw11/Dw5. One of these (RGT1) was shown to be functionally normal in DRw11-restricted alloreactive and antigen-specific systems. The specificity of MP2 was confirmed in functional assays: it was able to inhibit the recognition of DR1 and DRw15 but not DRw11 by alloreactive T-cell clones. Previously reported sequence data show that the beta chain of DRw11 differs from all other DR and DQ beta chains at position 58 by a glutamic acid for alanine substitution. This may account for the inability of DRw11/Dw5 to be recognized by MP2. The data emphasize the value of transfectants in defining precisely the allelic and chain specificity of anti-major-histocompatibility-complex (MHC) antibodies, and illustrate the influence that inaccessible residues can have on the conformation of MHC molecules.