Bleomycin, a chemotherapeutic agent, can cause pulmonary fibrosis in humans and is commonly used to induce experimental pulmonary fibrosis in rodents. In cell culture, bleomycin causes single- and double-stranded DNA breaks and produces reactive oxidative species, both of which require iron (Fe(2+)) and O(2). The mechanism of bleomycin-induced apoptosis is controversial due to its complexity. We investigated bleomycin apoptotic signaling events in primary pulmonary endothelial cells. Time course experiments revealed that bleomycin induced apoptosis within 4 h. Caspase-8, the initiator caspase for the extrinsic pathway, was activated within 2 h and preceded activation of the effector caspases-3 and -6 (4 h). Caspase-9, the initiator of the intrinsic pathway and release of cytochrome c from the mitochondria were not detected at these time points. Bleomycin induced the expression of Bcl-2 and Bcl-x(L), Bcl-2 family member proteins that protect cells from the mitochondria-dependent intrinsic apoptosis. Real-time quantitative RT-PCR results demonstrated that, at 4-8 h, bleomycin induced expression of TNF and TNF receptor family genes known to induce the extrinsic apoptotic pathway. Silencing of the death receptor adaptor protein Fas-associated death domain by short interfering RNA significantly reduced bleomycin-induced apoptosis. Apoptosis was also abrogated by caspase-8 inhibition, but only slightly reduced by caspase-3 inhibition. Together, these data suggest that bleomycin initiates apoptosis via the extrinsic pathway.