Signaling by the transcription factor nuclear factor kappa B (NF-κB) involves its release from inhibitor kappa B (IκB) in the cytosol, followed by translocation into the nucleus. NF-κB regulation of IκBα transcription represents a delayed negative feedback loop that drives oscillations in NF-κB translocation. Single-cell time-lapse imaging and computational modeling of NF-κB (RelA) localization showed asynchronous oscillations following cell stimulation that decreased in frequency with increased IκBα transcription. Transcription of target genes depended on oscillation persistence, involving cycles of RelA phosphorylation and dephosphorylation. The functional consequences of NF-κB signaling may thus depend on number, period, and amplitude of oscillations.