The HpxO enzyme from Klebsiella pneumoniae was recently proposed, on the basis of genetic studies, to catalyze the hydroxylation of uric acid to 5-hydroxyisourate as part of the purine catabolic pathway. Its primary sequence suggests that the HpxO catalytic activity depends on a flavin cofactor (FAD), contrasting with all previously studied urate oxidase enzymes, which have no cofactor requirement. Here we demonstrate biochemically that HpxO is an FAD-dependent urate oxidase. Our data are consistent with the proposal that HpxO-bound flavin hydroperoxide is the hydroxylating species. These results confirm the existence of a novel mechanistic paradigm in purine catabolism.