Hypoxia causes up-regulation and activation of xanthine dehydrogenase/xanthine oxidase (XDH/XO) in vitro and in the lungs in vivo. This up-regulation, and the likely corresponding production of reactive oxygen species, may underlie the pathogenesis of an array of disorders. Thus, compounds that prevent hypoxia-induced increase in XDH/XO activity may provide a therapeutic strategy in such disorders. The antioxidant properties of estrogens have been demonstrated in several studies. However, the effect of these compounds on XDH/XO has not been explored previously. The aim of this study was to investigate the effects of estrogen on hypoxia-induced increase in XDH/XO activity. Rat pulmonary artery microvascular endothelial cells were exposed to normoxia or hypoxia in the presence or absence of 17beta- or 17alpha-estradiol. The XDH/XO enzyme and gene promoter activities were measured in different groups of cells. Hypoxia caused a twofold increase in XDH/XO enzymatic and promoter activity. Either of the estradiol stereoisomers prevented the hypoxia-induced increase in XDH/XO enzymatic activity, but not the promoter activity. ICI 182,780, an antagonist of the estrogen receptor, failed to block the inhibitory effect of estradiol on XDH/XO. In conclusion, 17alpha- and 17beta-estradiol modulate the hypoxia-induced regulation of XDH/XO activity at a posttranscriptional level by a receptor-independent mechanism.