RelA and RelB belong to the nuclear factor-kappaB (NF-kappaB-Rel) transcription factor family. Both proteins are structurally and functionally related, but their intracellular and tissue distributions are different. In resting cells, RelB is found mostly in the nucleus, whereas RelA is sequestered in the cytosol by protein inhibitors, among which IkappaBalpha is the dominant form in lymphocytes. Upon cellular activation IkappaBalpha is proteolyzed, allowing RelA dimers to enter the nucleus and activate target genes. To study the selectivity of gene regulation by RelA and RelB, we generated T cell lines stably expressing a dominant negative mutant of IkappaBalpha. We show that selective inhibition of RelA-NF-kappaB decreased induction of NFKB1, interleukin-2, and interleukin-2Ralpha genes but not c-myc. Transcription driven by the IkappaBalpha promoter was blocked by the transgenic IkappaBalpha; however, wild type IkappaBalpha was expressed in the transgenic cell clones but with much slower kinetics than that in control cells. Wild type IkappaBalpha expression was concomitant with RelB up-regulation, suggesting that RelB could be involved in transcription of IkappaBalpha through binding to an alternative site. These results indicate that RelB and RelA have both distinct and overlapping effects on gene expression.