The specificity of three commonly used monoclonal antibodies (MoAbs) reacting with human class II histocompatibility antigens, was analyzed to determine whether these MoAbs would distinguish between HLA-DP, DQ, and DR in a large number of haplotypes. The reactivity of these MoAbs (L243, Anti-Leu 10, and B7/21) was compared by serial immunoprecipitation of class II antigens from 11 B-cell lines. The cell lines examined expressed a total of five DP, three DQ, and nine DR types, which together represent most of the well-defined class II specificities. This is the first demonstration that one of these antibodies, B7/21. binds to at least five DP specificities, and does not bind to DR or DQ molecules as defined by reactivity with the two other MoAbs. Within the scope of these experiments, the B7/21 antibody was shown to react with a monomorphic DP determinant. A variant clone of the B7/21 hybridoma was isolated that secretes IgG1 antibody with the same specificity as the original IgG3 antibody. The two other antibodies studied have been previously shown to react with DR molecules (L243) or DQ molecules (Anti-Leu 10). Here, their lack of cross-reaction with DP molecules is demonstrated. Thus, each of the three MoAbs reacts exclusively with a distinct class II molecule in all haplotypes studied, and therefore should be useful for comparing the independent expression and function of DP, DQ, and DR molecules.