Rofecoxib is a potent and highly selective cyclooxygenase-2 inhibitor used for the treatment of osteoarthritis and pain. Following administration of [4-(14)C]rofecoxib to intact rats, the plasma C(max) (at approximately 1 h) was followed by a secondary C(max) (at approximately 10 h), which was not observed in bile duct-cannulated rats. Following administration of [4-(14)C]5-hydroxyrofecoxib to intact or bile duct-cannulated rats, radiolabeled rofecoxib was detected in plasma, and once again a secondary C(max) for rofecoxib was observed (at approximately 10 h), which occurred only in the intact animals. These results indicate that reversible metabolism of rofecoxib to 5-hydroxyrofecoxib occurs in the rat and that the process is dependent upon an uninterrupted bile flow. Studies on the contents of the gastrointestinal tract of rats showed that conversion of 5-hydroxyrofecoxib to parent compound occurs largely in the lower intestine. Treatment of rats with [5-(18)O]5-hydroxyrofecoxib, followed by liquid chromatography-tandem mass spectrometry analyses of plasma samples, confirmed that 5-hydroxyrofecoxib undergoes metabolism to the parent drug, yielding [1-(18)O]rofecoxib, [2-(18)O]rofecoxib, and unlabeled rofecoxib. Similarly, treatment with [1,2-(18)O(2)]rofecoxib afforded the same three isotopic variants of rofecoxib. These findings are consistent with a metabolic sequence involving 5-hydroxylation of rofecoxib, biliary elimination of the corresponding glucuronide, and deconjugation of the glucuronide in the lower gastrointestinal tract. Reduction of the 5-hydroxyrofecoxib thus liberated yields a hydroxyacid that cyclizes spontaneously to regenerate rofecoxib, which is reabsorbed and enters the systemic circulation. This sequence represents a novel form of enterohepatic recycling and reflects the susceptibility of 5-hydroxyrofecoxib, as well as rofecoxib itself, to reversible 2-furanone ring opening under in vivo conditions.