The Fas/Fas ligand (FasL) apoptotic pathway has been shown to be involved in bleomycin-induced lung fibrosis. We examined the hypothesis that myofibroblasts from fibrotic lungs possess a cytotoxic phenotype that causes apoptosis of epithelial cells via the Fas/FasL pathway. We show in vivo epithelial cell apoptosis and associated upregulation of Fas and apoptotic Fas pathway genes in epithelial cells of lungs with bleomycin-induced fibrosis. In addition, we show that FasL surface molecules are overexpressed on alpha-SMA-positive cells in mice with bleomycin-induced fibrosis, and in humans with idiopathic pulmonary fibrosis. This enables the molecules to kill Fas-positive epithelial cells. In contrast, FasL-deficient myofibroblasts lose this myofibroblast cytotoxic phenotype, both in vivo and in vitro. In vivo, there was no bleomycin-induced epithelial cell apoptosis, as assessed by specific M30 staining in chimeric FasL-deficient mice that lacked FasL-positive myofibroblasts. In vitro, FasL-positive, but not FasL-negative myofibroblasts, induce mouse lung epithelial cell apoptosis. Thus myofibroblast cytotoxicity may underlie the absence of re-epithelialization, resulting in persistent lung fibrosis.