TNF-alpha is a potent proinflammatory cytokine that regulates immune and inflammatory responses and programmed cell death. TNF-alpha stimulation causes nuclear translocation of several NF-kappaB dimers, including RelA/p50 and RelB/p50. However, contrary to RelA, RelB entering the nucleus in response to TNF-alpha cannot bind to DNA in mouse embryonic fibroblasts, strongly suggesting that RelB DNA-binding activity is modulated by additional nuclear mechanisms. Here, we demonstrate that TNF-alpha promotes the association of RelA with RelB in the nucleus and that TNF-alpha-induced RelA/RelB heterodimers do not bind to kappaB sites. Remarkably, we show that RelA serine-276, the phosphorylation of which is induced by TNF receptor ligation, is crucial for RelA/RelB complex formation and subsequent inhibition of RelB DNA binding. In the absence of RelA phosphorylation on serine-276, TNF-alpha stimulation leads to a strong increase in the expression of endogenous NF-kappaB-responsive genes, such as Bcl-xL, whose transcriptional up-regulation is mainly controlled by RelB. Our findings demonstrate that RelA has a major regulatory role serving to dampen RelB activity in response to TNF-alpha and define a previously unrecognized mechanism that represents an essential step leading to selective NF-kappaB target gene expression.