The role of ubiquitylation in signal-induced activation of nuclear factor -kB (NF-kB) has been well established, while its involvement in maintaining NF-kB basal activity is less certain. Recent evidences demonstrate that in unstimulated cells, NF-kB homeostasis is actually the result of opposing forces: pro-activating activity of the IkB Kinase (IKK) and inhibitory activity of the Inhibitor of -kB (IkB) proteins. It is well known that endogenous deubiquitylating mechanisms are less effective on Ub motifs containing UbG76A. Here, we show that overexpression of a ubiquitin (Ub) G76A mutant leads to persistent activation of the IKK/NF-kB pathway in the absence of extra-cellular stimuli. In contrast, no effects on NF-kB activation were observed upon expression of UbK48R and UbK63R mutants, which are known to impair elongation of Lys48- and Lys63-linked poly-ubiquitin chains, respectively. Overall, these findings indicate that under basal conditions, the rate of de-ubiquitylation, rather than that of substrate ubiquitylation, is critical for the maintenance of appropriate levels of IKK/NF-kB activity.