Pulmonary epithelial cells are exposed to repetitive deformation during physiological breathing and mechanical ventilation. Such deformation may influence pulmonary growth, development, and barotrauma. Although deformation stimulates proliferation and activates extracellular-regulated kinases (ERK1/2) in human pulmonary epithelial H441 cells, the upstream mechanosensors that induce ERK activation are poorly understood. We investigated whether c-Src or Focal Adhesion Kinase (FAK) mediate cyclic mechanical strain-induced ERK1/2 activation and proliferation in human pulmonary epithelial (NCI-H441) cells. The H441 and A549 cells were grown on collagen I-precoated membranes and were subjected to an average 10% cyclic mechanical strain at 20 cycles/minute. Cyclic strain activated Src within 2 minutes by increasing phosphorylation at Tyr⁴¹⁸ followed by rapid phosphorylation of FAK at Tyr³⁹⁷ and Tyr⁵⁷⁶, and ERK1/2 at Thr²⁰²/Tyr²⁰⁴ (n=5, p<0.05). Twenty four (A549 cells) and 24-72 hours (H441 cells) of cyclic mechanical strain increased cell numbers compared with static culture. Twenty four hours of cyclic strain also increased H441 FAK, Src, and ERK phosphorylation without affecting total FAK, Src, or ERK protein. The mitogenic effect was blocked by Src (10 micromole PP2 or short interfering RNA targeted to Src) or MEK (50 micromole PD098059) inhibition. PP2 also blocked strain-induced phosphorylation of FAK-Tyr⁵⁷⁶, and ERK-Thr²⁰²/Tyr²⁰⁴ but not FAK-Tyr³⁹⁷ phosphorylation. Reducing FAK by FAK targeted short interfering RNA blocked mechanical strain-induced mitogenicity and significantly attenuated strain induced ERK activation, but not strain-induced Src phosphorylation. Taken together, these results suggest that repetitive deformation induced by ventilation supports pulmonary epithelial proliferation by a pathway involving Src, FAK, and then ERK signaling.