The objective of this study was to further investigate the metabolism of the 5alpha-reductase inhibitor, finasteride, and to identify previously unknown phase I and phase II metabolites in vitro and in vivo in human bile and urine. Healthy volunteers were given 5 mg of finasteride, directly to the intestine, and bile and urine were collected for 3 and 24 h, respectively. A single-pass perfusion technique, Loc-I-Gut, was used for drug administration and bile collection from the proximal jejunum, distal to papilla of Vater. Incubations with human liver microsomes/S9 fractions and different cofactors were performed with finasteride and the previously known metabolites, omega-hydroxy finasteride (M1) and finasteride-omega-oic acid (M3). Liquid chromatography coupled to triple quadrupole mass spectrometry (MS) with positive/negative electrospray ionization and ion trap with MS(n) measurements were used for structural investigations and identification of metabolites. Two hydroxy metabolites of finasteride, other than M1, and one intact hydroxy finasteride glucuronide were identified in vitro and in bile and urine. The glucuronide and at least one of the hydroxy metabolites were previously unidentified. M1 and M3 were glucuronidated in vitro by specific human UDP-glucuronosyltransferases, UGT1A4 and UGT1A3, respectively. M1 glucuronide was not identified in vivo, and M3 glucuronide, an acyl glucuronide, was present in low amounts in bile from a few individuals. In conclusion, previously undescribed metabolites were identified, in vitro and in human urine and bile. Bile collection using the Loc-I-Gut technique followed by sensitive mass spectrometry analysis led to the discovery of novel, both phase I and phase II, finasteride metabolites in human bile.