The signaling pathways by which mechanical forces modulate fetal lung development remain largely unknown. In the present study, we tested the hypothesis that strain-induced fetal type II cell differentiation is mediated via the cAMP signaling pathway. Freshly isolated E19 fetal type II epithelial cells were cultured on collagen-coated silastic membranes and exposed to mechanical strain for varying intervals, to simulate mechanical forces during lung development. Unstretched samples were used as controls. Mechanical strain activated heterotrimeric G-protein αssubunit, cAMP, and the transcription factor cAMP response element binding protein (CREB). Incubation of E19 cells with the PKA inhibitor H-89 significantly decreased strain-induced CREB phosphorylation. Moreover, adenylate cyclase 5 and CREB genes were also mechanically induced. In contrast, components of the PKA-independent (Epac) pathway, including Rap-1 or B-Raf, were not phosphorylated by strain. The addition of forskolin or dibutyryl cAMP to unstretched E19 monolayers markedly upregulated expression of the type II cell differentiation marker surfactant protein C, whereas the Epac agonist 8-pCPT-2′- O-Me-cAMP had no effect. Furthermore, incubation of E19 cells with the PKA inhibitor Rp-2′- O-monobutyryladenosine 3′,5′-cyclic monophosphorothioate or transient transfection with plasmid DNA containing a PKA inhibitor expression vector significantly decreased strain-induced surfactant protein C mRNA expression. In conclusion, these studies indicate that the cAMP-PKA-dependent signaling pathway is activated by force in fetal type II cells and participates in strain-induced fetal type II cell differentiation.