Reactive oxygen species have been reported to be involved in the airway inflammatory process of chronic obstructive pulmonary disease (COPD). The aim of this study was to quantify the activity of xanthine oxidase (XO), which generates a potent radical superoxide anion in COPD airways. Thirteen stable COPD patients and 10 healthy subjects participated in this study. We collected the epithelial lining fluid using a newly developed microsampling technique, and quantified of cytokines responsible for the XO gene upregulation. The XO activity was significantly increased in COPD patients compared with that in healthy subjects. A significant negative correlation was found between the XO activity and the %FEV1 values. The level of tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma in COPD patients was significantly higher than that in healthy subjects. Both the amount of tumor necrosis factor-alpha and interleukin-1beta were significantly correlated with the degree of XO activity. These results suggest that the XO activity is increased in COPD airways, possibly due to its gene upregulation by proinflammatory cytokines. Because the XO activity was significantly correlated with the degree of airway obstruction, these cytokine-XO production pathways may play a key role in the inflammation of COPD.