The chemotherapeutic agent bleomycin induces pulmonary fibrosis through the generation of reactive oxygen species (ROS), which are thought to contribute to cellular damage and pulmonary injury. We hypothesized that bleomycin activates oxidative stress response pathways and regulates cellular glutathione (GSH). Bovine pulmonary artery endothelial cells exposed to bleomycin exhibit growth arrest and increased cellular GSH content. -Glutamylcysteine synthetase (-GCS) controls the key regulatory step in GSH synthesis, and Northern blots indicate that the -GCS catalytic subunit [-GCS heavy chain (-GCS_h)] is upregulated by bleomycin within 3 h. The promoter for human -GCS_h contains consensus sites for nuclear factor-B (NF-B) and the antioxidant response element (ARE), both of which are activated in response to oxidative stress. Electrophoretic mobility shift assays show that bleomycin activates the transcription factor NF-B as well as the ARE-binding factors Nrf-1 and -2. Nrf-1 and -2 activation by bleomycin is inhibited by the ROS quenching agent N-acetylcysteine (NAC), but not by U-0126, a MEK1/2 inhibitor that blocks bleomycin-induced MAPK activation. In contrast, NF-B activation by bleomycin is inhibited by U-0126, but not by NAC. NAC and U-0126 both inhibit bleomycin-induced upregulation of -GCS expression. These data suggest that bleomycin can activate oxidative stress response pathways and upregulate cellular GSH.