Neutrophils are among the first circulating leukocytes involved in acute inflammatory processes. Transcription factor NF-κB plays a key role in the inflammatory response, regulating the expression of proinflammatory and anti-apoptotic genes. Recently we have shown that human neutrophils contain a significant amount of NF-κB inhibitor, IκBα, in the nucleus of unstimulated cells. The present objective was to examine the mechanisms controlling the nuclear content of IκBα in human neutrophils and to determine whether increased accumulation of IκBα in the nucleus is associated with increased neutrophil apoptosis. We show for the first time that neutrophil stimulation with pro-inflammatory signals results in degradation of IκBα that occurs in both cytoplasm and nucleus. Prolonged (2-h) stimulation with TNF and LPS induces resynthesis of IκBα that is again translocated to the nucleus in human neutrophils, but not in monocytic cells. Leptomycin B, a specific inhibitor of nuclear export, increases nuclear accumulation of IκBα in stimulated neutrophils by blocking the IκBα nuclear export, and this is associated with inhibition of NF-κB activity, induction of caspase-3 activation, and apoptosis. Based on our data we present a new model of NF-κB regulation in human neutrophils by nuclear IκBα. Our results demonstrate that the NF-κB activity in human neutrophils is regulated by mechanisms clearly different from those in monocytes and other human cells and suggest that the increased nuclear content of IκBα in human neutrophils might represent one of the underlying mechanisms for the increased apoptosis in these cells.