HLA-DRB1 and -DRB3 are two genes encoding two distinct HLA-DR beta chains in the DRw52 family of haplotypes (DR3, DR5, DR13, and DR14). These beta chains determine the structural and functional identity of the two kinds of HLA-DR molecules expressed. The highly polymorphic HLA-DRB1 locus is always expressed at a higher level than the HLA-DRB3 locus, and functional assays indicate that the proximal promoter of DRB1 is indeed more active than that of DRB3. The DNA sequence of the two promoters in nine different DRw52 haplotypes has revealed a stricking allelic conservation as well as characteristic, isotype-specific, conserved-sequence motifs. These isotype-specific differences concern the functionally essential X and Y box motifs of HLA class II promoters, and they do indeed affect binding of specific nuclear factors to the X and Y boxes of DRB1 or DRB3 promoters. Finally, analysis of the activity of various normal and mutated DRB1 or DRB3 promoters indicates that the X box region of these promoters plays a dominant role in controlling the relative levels of HLA-DRB1 and -DRB3 gene expression.