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Summary Nuclear factor (NF)-κB controls the transcriptional response to inflammatory signals by translocating into the nucleus, but we lack a single-cell characterization of the resulting transcription dynamics. Here we show that upon tumor necrosis factor (TNF)-α transcription of NF-κB target genes is heterogeneous in individual cells but results in an average nascent transcription profile that is prompt (i.e., occurs almost immediately) and sharp (i.e., increases and decreases rapidly) compared with NF-κB nuclear localization. Using an NF-κB-controlled MS2 reporter we show that the single-cell nascent transcription is more heterogeneous than NF-κB translocation dynamics, with a fraction of synchronized “first responders” that shape the average transcriptional profile and are more prone to respond to multiple TNF-α stimulations. A mathematical model combining NF-κB-mediated gene activation and a gene refractory state is able to reproduce these features. Our work shows how the expression of target genes induced by transcriptional activators can be heterogeneous across single cells and yet time resolved on average., Graphical Abstract, Highlights • Nascent transcription upon TNF-α is heterogeneous, with a subset of “first responders” • The average nascent transcription is prompt and sharper than NF-κB response • First responders do not depend on NF-κB dynamics and respond more to pulsed stimuli • A model including NF-κB and a gene refractory state reproduces these observations, Biological Sciences; Molecular Biology; Immunology; Biophysics; Systems Biology