The Nuclear Factor kappa B (NF-κB) transcription factor regulates cellular stress responses and the immune response to infection. NF-κB activation results in oscillations in nuclear NF-κB abundance. To define the function of these oscillations, we treated cells with repeated short pulses of tumor necrosis factor alpha (TNFα) at various intervals to mimic pulsatile inflammatory signals. At all pulse intervals analyzed, we observed synchronous cycles of NF-κB nuclear translocation. Lower frequency stimulations gave repeated full-amplitude translocations, whereas higher frequency pulses, gave reduced translocation, indicating a failure to reset. Deterministic and stochastic mathematical models predicted how negative feedback loops regulate both the resetting of the system and cellular heterogeneity. Altering the stimulation intervals gave different patterns of NF-κB-dependent gene expression, supporting a functional role for oscillation frequency.Eukaryotic cells interpret multiple signals to coordinate the activity of transcription factors, which modulate the expression of target genes. NF-κB signaling in many mammalian cell types regulates responses to pathogens and stresses (1). NF-κB, most commonly comprising a dimer of RelA and p50, is bound in the cytoplasm of unstimulated cells by inhibitor kappa B (IκB) proteins. Stimulation by cytokines such as TNFα activates the inhibitor kappa B kinase (IKK) complex that phosphorylates IκB proteins, leading to IκB degradation and NF-κB translocation into the nucleus. Activated NF-κB regulates transcription from promoter regions of approximately 300 genes, including those encoding cytokines and several NF-κB family members which can feedback to regulate the system (2). Signaling through NF-κB can regulate diverse cellular outcomes including cell death or division (3). How such a diversity of responses is generated has remained unclear.Real-time fluorescence imaging and mathematical modeling have shown that the activity of the NF-κB system can be oscillatory (4). This raised the possibility that, as with calcium (5) (7)). In contrast to the conclusions of other reports (9, 10), we observed oscillations in the translocation of RelA-dsRedxp fusion protein in single transiently transfected MEFs (Fig. 1, C, D and E). These data (as well as bulk cell chromatin immunoprecipitation (ChIP) assays, Fig. S1), suggest that oscillations are a normal response to TNFα stimulation.In an inflammatory tissue, cells receive pulsatile signals such as TNFα from neighboring cells (11,12). To mimic this, we exposed cells to 5 min pulses of TNFα at various intervals, followed by wash-off. When stimulated at 200-min intervals, RelA-dsRedxp fusion protein expressed in SK-N-AS cells showed synchronous translocations from the cytoplasm to the nucleus and back of equal magnitude in response to each successive pulse (Fig. 2, A and B and fig. S4). In contrast, whereas stimulation at 100 or 60 min intervals also caused synchronous cell responses, there was significant reduction in the magnitude ...