Increased extracellular matrix (ECM) deposition and airway smooth muscle (ASM) mass are major contributors to airway remodeling in asthma. Increased deposition of the ECM protein collagen I is not only observed beneath the epithelium, but also surrounding the asthmatic ASM bundle. Recently, we have demonstrated that collagen I induces a proliferative, hypocontractile ASM phenotype. Little is known, however, on the signalling pathways involved. Using bovine tracheal smooth muscle (BTSM), we now investigated the role of focal adhesion kinase (FAK) and downstream signalling pathways in collagen I-induced phenotype modulation. Phosphorylation of FAK was increased during adhesion to uncoated and collagen I-coated plastic culture dishes, without differences between the matrices. No differences between cellular adhesion were found either. Inhibition of FAK activity, by overexpression of the FAK deletion mutants FAT (focal adhesion targeting domain) and FRNK (FAK-related non-kinase), attenuated adhesion. After attachment, FAK phosphorylation was time-dependently increased in cells cultured on collagen I, whereas no activation was found on the uncoated plastic matrix. In addition, collagen I time- and concentration-dependently increased BTSM cell proliferation, which was inhibited by FAT and FRNK. Collagen I-induced proliferation was also concentration-dependently inhibited by the pharmacological FAK Inhibitor PF-573,228 (FAK inhibitor II) at concentrations that were specific for FAK (IC50=65±16 nM). In addition, the induction of a hypocontractile phenotype by collagen I was inhibited by this compound. Specific pharmacological inhibitors of p38 MAPK (SB203580, 10 mM) and Src-kinase (PP2, 10 mM) fully inhibited collagen I-induced proliferation as well, whereas partial inhibition was observed by inhibition of PI3-kinase (LY294002, 10 mM) and MEK (U0126, 3 mM). Inhibition of cell proliferation by the inhibitors mentioned was associated with attenuation of the collagen I-induced hypocontractility. Collectively, the results indicate that induction of a proliferative, hypocontractile ASM phenotype by collagen I is mediated by FAK and by p38 MAPK, MEK, PI3-kinase and Src-mediated signaling pathways downstream of FAK.