Labeled cDNA probes highly enriched for B-cell-specific (B-T) and T-suppressor-cell-specific (Ts-B) sequences were used to screen a set of genomic cosmid clones spanning 230 kilobases of the murine immune response (I) region. With the B-cell derived probe, four I-region genes were detectable (A beta, E beta, E beta 2, and E alpha), as well as an additional (fifth) region of hybridization. The T-cell probe, prepared from a putative I-J positive suppressor cell hybridoma, was negative in a parallel experiment. A genomic fragment corresponding to the new region of hybridization seen with the B-cell cDNA probe identified a discrete mRNA species in RNA blotting analysis that had a pattern of expression strikingly similar to E alpha mRNA in a variety of lymphoid tumor lines. This fragment was also used to isolate cDNA clones from a library highly enriched (20-40 times) for B-cell-specific sequences (selected cDNA cloning). DNA sequence analysis of one of these cDNA clones indicates that this gene encodes the A alpha molecule of the d haplotype (Ad alpha). These findings establish that the order of the class II genes in the Ia complex is as follows: centromere-A beta-A alpha-E beta-E beta 2-E alpha.