The expression of many metazoan genes is regulated through controlled release of RNA polymerase II (Pol II) that has paused during early transcription elongation. Pausing is highly enriched at genes in stimulus-responsive pathways, where it has been proposed to poise downstream targets for rapid gene activation. However, whether this represents the major function of pausing in these pathways remains to be determined. To address this question, we analyzed pausing within several stimulus-responsive networks in Drosophila and discovered that paused Pol II is much more prevalent at genes encoding components and regulators of signal transduction cascades than at inducible downstream targets. Within immune-responsive pathways, we found that pausing maintains basal expression of critical network hubs, including the key NF-κB transcription factor that triggers gene activation. Accordingly, loss of pausing through knockdown of the pause-inducing factor NELF leads to broadly attenuated immune gene activation. Investigation of murine embryonic stem cells revealed that pausing is similarly widespread at genes encoding signaling components that regulate self-renewal, particularly within the MAPK/ERK pathway. We conclude that the role of pausing goes well beyond poising-inducible genes for activation and propose that the primary function of paused Pol II is to establish basal activity of signal-responsive networks.