Raltegravir is a potent human immunodeficiency virus 1 (HIV-1) integrase strand transfer inhibitor that is being developed as a novel anti-AIDS drug. The absorption, metabolism, and excretion of raltegravir were studied in healthy volunteers after a single oral dose of 200 mg (200 microCi) of [(14)C]raltegravir. Plasma, urine, and fecal samples were collected at specified intervals up to 240 h postdose, and the samples were analyzed for total radioactivity, parent compound, and metabolites. Radioactivity was eliminated in substantial amounts in both urine (32%) and feces (51%). The elimination of radioactivity was rapid, since the majority of the recovered dose was attributable to samples collected through 24 h. In extracts of urine, two components were detected and were identified as raltegravir and the glucuronide of raltegravir (M2), and each accounted for 9% and 23% of the dose recovered in urine, respectively. Only a single radioactive peak, which was identified as raltegravir, was detected in fecal extracts; raltegravir in feces is believed to be derived, at least in part, from the hydrolysis of M2 secreted in bile, as demonstrated in rats. The major entity in plasma was raltegravir, which represented 70% of the total radioactivity, with the remaining radioactivity accounted for by M2. Studies using cDNA-expressed UDP-glucuronosyltransferases (UGTs), form-selective chemical inhibitors, and correlation analysis indicated that UGT1A1 was the main UGT isoform responsible for the formation of M2. Collectively, the data indicate that the major mechanism of clearance of raltegravir in humans is UGT1A1-mediated glucuronidation.