Tumor necrosis factor (TNF)-α is a potent inflammatory cytokine implicated in the exacerbation of asthma. Chronic exposure to TNF-α has been reported to induce G protein-coupled receptor 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 G protein-coupled receptor signals, is an important second messenger involved in the regulation of the airway muscle proliferation, migration, and tone. In other cell types, TNF-α 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-α in the presence or absence of inhibitors of prostaglandin signaling, protein kinases, or Gi proteins. TNF-α caused a significant dose- (1–10 ng/ml) and time-dependent (24 and 48 h) 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 unaffected by pertussis toxin. TNF-α also increased phosphorylation of Ser338 on raf-1 kinase, indicative of activation. IL-1β 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-α transactivation of the EGF receptor, with subsequent raf-1 kinase-mediated activation of adenylyl cyclase.