The present study determined the effect of hypoxia on xanthine dehydrogenase (XDH) and xanthine oxidase (XO) activity and gene and protein expression in cultured bovine aortic endothelial cells (BAEC). BAEC were exposed to hypoxia (3% O2) or anoxia (0% O2) for 24 or 48 h and to 24 h of hypoxia followed by 24 h of reoxygenation. Hypoxia- and anoxia-exposed BAEC demonstrated a greater than twofold increase in XDH/XO activity at 24 and 48 h compared with timed controls. Hypoxic cells that were subsequently reoxygenated in 21% O2 also demonstrated a similar increase in XDH/XO activity vs. timed controls. No differences were seen in mRNA levels at any time point. Similarly, no difference was noted in XDH/XO protein expression after hypoxic exposure, as determined by Western blot analysis. The increase in XDH/XO activity was not prevented by cyclohexamide, indicating that protein synthesis was not required. Thus the increased XDH/XO activity observed in response to hypoxia in the present study was due to posttranslational modulation of the enzyme.