LPS and selected cytokines upregulate xanthine dehydrogenase/xanthine oxidase (XDH/XO) in cellular systems. However, the effect of these factors on in vivo XDH/XO expression, and their contribution to lung injury, are poorly understood. Rats were exposed to normoxia or hypoxia for 24 h after treatment with LPS (1 mg/kg) and IL-1β (100 μg/kg) or sterile saline. Lungs were then harvested for measurement of XDH/XO enzymatic activity and gene expression, and pulmonary edema was assessed by measurement of the wet/dry lung weight ratio (W/D). Although treatment with LPS + IL-1β or hypoxia independently produced a 2-fold elevation (p < 0.05 versus exposure to normoxia and treatment with saline) in lung XDH/XO activity and mRNA, the combination of LPS + IL-1β and hypoxia caused a 4- and 3.5-fold increase in these values, respectively. XDH/XO protein expression was increased 2-fold by hypoxia alone and 1.3-fold by treatment with LPS + IL-1β alone or combination treatment. Compared with normoxic lungs, W/D was significantly increased by exposure to hypoxia, LPS + IL-1β, or combination treatment. This increase was prevented by treatment of the animals with tungsten, which abrogated lung XDH/XO activity. In conclusion, LPS, IL-1β, and hypoxia significantly upregulate lung XDH/XO expression in vivo. The present data support a role for this enzyme in the pathogenesis of acute lung injury.