Tumor necrosis factor (TNF) is a potent inflammatory cytokine implicated in the exacerbation of asthma. Chronic exposure to TNF has been reported to induce G protein coupled receptors (GPCR) desensitization but adenylyl cyclase sensitization in airway smooth muscle cells by an unknown mechanism. Cyclic AMP, which is synthesized by adenylyl cyclases in response to GPCR signals, is an important second messenger involved in the regulation of the airway muscle proliferation, migration and tone. In other cell types TNF receptors transactivate the EGF receptor which activates raf-1 kinase. Further studies in transfected cells show that raf-1 kinase can phosphorylate and activate some isoforms of adenylyl cyclase. Cultured human airway smooth muscle cells were treated with TNF in the presence or absence of inhibitors of prostaglandin signaling, protein kinases or G_i proteins. TNF caused a significant dose (1-10 ng/ml)- and time (24, 48hr)-dependent increase in forskolin-stimulated adenylyl cyclase activity which was abrogated by pretreatment with GW5074 (a raf-1 kinase inhibitor), was partially inhibited by an EGF receptor inhibitor but was uneffected by pertussis toxin. TNF also increased phosphorylation of Ser³³⁸ on raf-1 kinase indicative of activation. IL-1 and EGF sensitization of adenylyl cyclase activity was also sensitive to raf-1 kinase inhibition by GW5074. Taken together, these studies link two signaling pathways not previously characterized in human airway smooth muscle cells: TNF transactivation of the EGF receptor with subsequent raf-1 kinase mediated activation of adenylyl cyclase.