NF-kappaB activation is triggered by the degradation of inhibitory proteins, such as I kappaB-alpha. I kappaB-alpha levels are only transiently lowered since one gene activated by NF-kappaB is I kappaB-alpha. We found that I kappaB-alpha was replenished rapidly in a human colon cell line (HT-29), even in the presence of degradation-inducing phosphorylation (at serine-32). This finding lead us to hypothesize that posttranscriptional mechanisms were also in place to facilitate I kappaB-alpha replenishment. Expression of I kappaB-alpha from the constitutive, non-NF-kappaB regulated cytomegalovirus promoter in HT-29 cells showed that TNF-alpha or IL-1 beta treatment increased I kappaB-alpha levels in the absence of transcriptional activation. The TNF-alpha -induced increase in transgenic I kappaB-alpha appeared to result from the stabilization of newly synthesized I kappaB-alpha, since this increase was effectively preempted by a proteasome inhibitor (MG132) or by I kappaB-alpha stabilization through the deletion C-terminal destabilizing elements (without additive or synergistic effects). Analysis of a hepatoma cell line (Hepa 1-4C7) indicated that the I kappaB-alpha stabilization may be constitutive in these cells. NF-kappaB stimuli therefore appear to trigger negative feedback pathways in some cells that terminate a NF-kappaB response by increasing the stability of newly synthesized I kappaB-alpha.