The early response gene IEX-1 is involved in the regulation of cellular growth and survival, and its expression is related to stress-, growth-and death-inducing signals. Addressing the role of IEX-1 in the promotion of apoptosis, we investigated the effect of IEX-1 on nuclear factor-κB (NF-κB) activation. Stably transfected HEK-293 cells conditionally overexpressing IEX-1 exhibit decreased levels of NF-κB activity, either basal or TNFα induced, as shown by gel-shift and luciferase reporter gene assay. Furthermore, activated p65 accumulated in the nuclei of 293 cells to a lower degree, if IEX-1 expression was increased. This inhibited NF-κB activation was preceded by an altered turnover of IκBα and phospho-IκBα. In addition, IEX-1 expression also inhibited the activity of the 26S-proteasome, as shown by a fluorometric proteasome assay. Conversely, disruption of IEX-1 expression in 293 cells by stable transfection with specific anti-IEX-1 hammerhead ribozymes increased NF-κB activity, and accelerated the degradation of IκBα. Along with these opposite effects of IEX-1 expression and IEX-1 disruption on NF-κB activation, the sensitivity of 293 cells towards various apoptotic stimuli also changed. In contrast to ribozyme-transduced 293 cells that were significantly less sensitive to apoptosis, this sensitivity was enhanced if IEX-1 expression was increased. Our data suggest that IEX-1–itself an NF-κB target gene–inhibits the activation of this transcription factor, and hereby may counteract the antiapoptotic potential of NF-κB.