Monoclonal antibody IVD12 was used to isolate and characterize a human Ia molecule present on B cells that generally display DR4 or DR5 phenotypes. The specificity of binding of IVD12 to human peripheral blood B cells from 75 normal individuals and 19 homozygous human lymphoblastoid B cell lines was identical to the supertypic specificity MB3 previously defined. Furthermore, IVD12-reactivity was shown to segregate with HLA in three informative families. In each family, individuals positive for IVD12 binding were also positive for DR4 or DR5. Using IVD12, a molecule has been isolated from the homozygous cell line PRIESS (DR4/4) and has been shown by amino acid sequence analysis to be homologous to the murine I-A and human HLA-DS molecules. These findings suggest that the MB3 specificity is found on a molecule encoded by loci distinct from those loci which encode HLA-DR molecules. This molecule represents the third family of HLA-D region molecules isolated from the cell line PRIESS. Both HLA-DR and HLA-SB molecules from this cell line were previously shown by amino acid sequence analysis to be I-E-like but distinct from one another. Collectively, these data provide evidence that the HLA-D region contains at least six loci encoding distinct alpha and beta chains for the HLA-SB, HLA-DR, and HLA-DS molecules.