The molecular mechanisms that regulate nuclear NF-kappaB to determine whether the stimulation of this pathway has a pro- or anti-apoptotic effect on cells have yet to be fully defined. Nuclear compartmentalization is increasingly recognized as an important mechanism for regulating the activity of transcription-related proteins and modulating cell growth and death. We have investigated whether such compartmentalization serves as a mechanism for regulating NF-kappaB transcriptional activity. We demonstrate that the RelA component of NF-kappaB is sequestered in the nucleolus in response to the proapoptotic NF-kappaB stimuli aspirin, serum withdrawal, and UV-C radiation. In contrast, RelA is excluded from the nucleolus in response to the cytokines tumor necrosis factor and TRAIL. We identify an N-terminal motif of RelA that is essential for the nucleolar localization of the protein and show that deleting this motif inhibits the translocation of RelA from the nucleoplasm to the nucleolus. We demonstrate that the nucleolar accumulation of RelA is paralleled by a decrease in basal levels of NF-kappaB transcriptional activity and by apoptosis. Furthermore, we show that the retention of RelA in the nucleoplasm inhibits this decrease in NF-kappaB-driven transcription and blocks apoptosis induced by aspirin and UV-C radiation. This work identifies a novel cellular mechanism for regulating NF-kappaB-driven transcription and apoptosis, involving the nucleolar sequestration of a key NF-kappaB subunit. These data contribute to the understanding of the complexities of NF-kappaB function and have considerable relevance to cancer prevention and therapy.