The onset of parturition is associated with a number of proinflammatory mediators that are themselves regulated by the nuclear factor κB (NF-κB) family of transcription factors. In this context, we previously reported that the RelA NF-κB subunit represses transcription and mRNA expression of the proquiescent Gαs gene in human myometrial cells following stimulation with the proinflammatory cytokine TNF. In the present study, we initially defined the functional consequence of this on myometrial contractility. Here we show that, contrary to our initial expectations, TNF did not induce myometrial contractility but did inhibit the relaxation produced by the histone deacetylase inhibitor trichostatin A, an effect that in turn was abolished by the NF-κB inhibitor N(4)-[2-(4-phenoxyphenyl)ethyl]-4,6-quinazolinediamine. This result suggested a role for TNF in regulating Gαs expression via activating NF-κB and modifying histone acetylation associated with the promoter region of the gene. In this context, we show that the -837 to -618 region of the endogenous Gαs promoter is occupied by cAMP-response element-binding protein (CREB), Egr-1, and Sp1 transcription factors and that CREB-binding protein (CBP) transcriptional complexes form within this region where they induce histone acetylation, resulting in increased Gαs expression. TNF, acting via NF-κB, did not change the levels of CREB, Sp1, or Egr-1 binding to the Gαs promoter, but it induced a significant reduction in the level of CBP. This was associated with increased levels of histone deacetylase-1 and surprisingly an increase in H4K8 acetylation. The latter is discussed herein.