We have produced a monoclonal antibody detecting an HLA class II determinant absent from DR1,2,W6, and W10 (DQW1) homozygous B lymphoblastoid cell lines (BLCL). The antibody, SFR16-PI.2, immunoprecipitates molecules with electrophoretic mobilities of DQ (DS/DC) molecules from DR7 homozygous cell lines. SFR16-PI.2 binds more sets of class II molecules from internally labeled DR7 homozygous cell membranes than from externally labeled extracts from the same cell line. Depletion of DR molecules from internally-labeled membranes indicates that SFR16-PI.2, in addition to reacting with DQ molecules, also reacts with an epitope on a biosynthetic intermediate of DR molecules, which is lost on the mature DR molecule. SFR16-PI.2-reactive molecules were examined on a DR5 homozygous cell line, where they could be compared to those isolated by two other monoclonal antibodies with DQ specificity, namely IVD12 and Leu-10. All three monoclonal antibodies isolated two sets of DQ molecules with varying degrees of affinity for one of the molecules. The serological and biochemical data presented suggest that SFR16-PI.2 detects the alternate alleles of the locus encoding DQW1.