The mechanism regulating the differential expression of DR beta 1 and DR beta 4 proteins on the surfaces of cultured EBV-transformed DR7 cell lines was investigated at the level of gene transcription. Steady-state levels of mRNA coding for DR7 beta 1 and DR beta 4 proteins were evaluated with the use of locus-specific oligonucleotide probes and relative transcriptional activity at the DR7B1 and DRB4 gene loci was measured with nuclear run-on transcription assays. Steady-state levels of DR7B1 mRNA were found to be three- to nine-fold higher than those of DRB4 in DR7,Dw7 cell lines, but no mature steady-state DRB4 mRNA was detectable in a DR7,Dw11 cell line which shows no detectable DR beta 4 protein. Run-on transcription assays performed with nuclei isolated from DR7,Dw7 cells showed a five-fold excess of DRB1 over DRB4 transcriptional activity, but, surprisingly, no difference in activity at the two DRB loci in nuclei isolated from DR7,Dw11 cells. Both DRB mRNA were quite stable as indicated by actinomycin D time course experiments. Thus the differential expression of the DR7 beta 1 and DR beta 4 proteins in the DR7,Dw7 cell lines arises at the level of mRNA production; the lack of detectable DR beta 4 protein in the DR7,Dw11 cell line, however, appears to be due to a different mechanism in which transcribed DRB4 sequences fail to be processed into mature mRNA molecules.