The mechanism generally admitted for the bioactivation of the antithrombotic prodrug, prasugrel, 1c, is its two-step enzymatic conversion into a biologically active thiol metabolite. The first step is an esterase-catalyzed hydrolysis of its acetate function leading to a thiolactone metabolite 2c. The second step was described as a cytochrome P450 (P450)-dependent oxidative opening of the thiolactone ring of 2c, with intermediate formation of a reactive sulfenic acid metabolite that is eventually reduced to the corresponding active thiol 3c. This article describes a detailed study of the metabolism of 1c by human liver microsomes and human sera, with an analysis by HPLC-MS under conditions allowing a complete separation of the thiol metabolite isomers, after derivatization with 3'-methoxy phenacyl bromide. It shows that there are two competing metabolic pathways for the opening of the 2c thiolactone ring. The major one, which was previously described, results from a P450- and NADPH-dependent redox bioactivation of 2c and leads to 3c, two previously reported thiol diastereomers bearing an exocyclic double bond. It occurs with NADPH-supplemented human liver microsomes but not with human sera. The second one results from a hydrolysis of 2c and leads to an isomer of 3c, 3c endo, in which the double bond has migrated from an exocyclic to an endocyclic position in the piperidine ring. It occurs both with human liver microsomes and human sera, and does not require NADPH. However, it requires Ca(2+) and is inhibited by paraoxon, which suggests that it is catalyzed by a thioesterase such as PON-1. Chemical experiments have confirmed that hydrolytic opening of thiolactone 2c exclusively leads to derivatives of the endo thiol isomer 3c endo.