The chemotherapeutic agent bleomycin induces pulmonary fibrosis through the generation of reactive oxygen species (ROS). We hypothesized that bleomycin activates oxidative stress response pathways and regulates cellular glutathione (GSH). Bovine pulmonary artery endothelial cells (BPAEC) exposed to bleomycin exhibit growth arrest and increased cellular glutathione 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 30 min. The promoter for human -GCS_h contains consensus sites for NF-B and the antioxidant response element (ARE), two cis elements that are known to be activated in response to oxidative stress. Electrophoretic mobility shift assays (EMSA)show that within 30 min bleomycin activates the transcription factors NF-B, as well as the ARE-binding factors Nrf-1 and -2. Nrf-1 and -2 activation is inhibited by the ROS quenching agent N-acetylcysteine(NAC), but not by U0126, a MEK1/2 inhibitor which blocks bleomycin-induced MAPK activation. In contrast NF-B activation by bleomycin is inhibited by U0126 but not by NAC. NAC and U0126 both inhibit bleomycin-induced upregulation of -GCS_h expression. These data suggest that bleomycin can activate oxidative stress response pathways and upregulate GSH.