Urethane, which is used as an anesthetic for animal experiments, causes inflammation and cancer in the lung. BALB/c mice received 1 mg/g of urethane once a week for four consecutive weeks via intraperitoneal injections developed interstitial infiltration of inflammatory cells and tumors in the lung. However, the intracellular signaling events which urethane causes inflammation and cancer are largely unknown. Here we show that urethane caused overproduction of reactive oxygen species (ROS) in RAW 264.7 macrophages and A549 lung epithelial cells. Pretreatment of these cells with the antioxidant N-acetylcysteine attenuated the urethane-induced ROS production. Urethane increased heme oxygenase-1 expression to protect these cells from cytotoxicity caused by overproduced ROS. In addition, urethane activated extracellular signal-regulated kinase (ERK) in both cell types. Overall, our data imply that urethane stimulates ROS production and ERK activation in macrophages and lung epithelial cells, and the overproduced ROS and activated ERK may promote tumor formation in the lung.