IkappaB inhibitor proteins are the primary regulators of NF-kappaB. In contrast to the defined regulatory interplay between NF-kappaB and IkappaBalpha, much less is known regarding the regulation of IkappaBbeta by NF-kappaB. Here, we describe in detail the regulation of IkappaBbeta by RelA/p65. Using p65(-/-) fibroblasts, we show that IkappaBbeta is profoundly reduced in these cells, but not in other NF-kappaB subunit knockouts. This regulation prevails during embryonic and postnatal development in a tissue-specific manner. Significantly, in both p65(-/-) cells and tissues, IkappaBalpha is also reduced, but not nearly to the same extent as IkappaBbeta, thus highlighting the degree to which IkappaBbeta is dependent on p65. This dependence is based on the ability of p65 to stabilize IkappaBbeta protein from the 26S proteasome, a process mediated in large part through the p65 carboxyl terminus. Furthermore, IkappaBbeta was found to exist in both a basally phosphorylated and a hyperphosphorylated form. While the hyperphosphorylated form is less abundant, it is also more stable and less dependent on p65 and its carboxyl domain. Finally, we show that in p65(-/-) fibroblasts, expression of a proteolysis-resistant form of IkappaBbeta, but not IkappaBalpha, causes a severe growth defect associated with apoptosis. Based on these findings, we propose that tight control of IkappaBbeta protein by p65 is necessary for the maintenance of cellular homeostasis.