The metabolic fate of the organophosphorothioate-type insecticide chlorpyrifos (CP) in an acutely intoxicated 59 years old female was investigated by liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI-MS/MS) analysis of urine samples. Fifteen metabolites of CP and its bioactivated intermediate chlorpyrifos-oxon (CPO), respectively, of which only three have been described in man so far, were identified on the basis of characteristic MS/MS transitions, precursor/product ion and/or neutral loss scans, chlorine isotopomer patterns, and partly by synthesis of reference compounds and subsequent structure confirmation. Three distinct biotransformation routes of CP are proposed: (1) cleavage reactions at the aromatic phosphoester bond, (2) cleavage reactions at the alkyl phosphoester bonds, and (3) glutathione (GSH) dependent nucleophilic substitution of the 6-chlorine at the aromatic moiety. Route (2) has not been reported in humans before and (3) is a hitherto completely unknown scheme of CP metabolism. Urinary markers of the latter were chiefly cysteine S-conjugates of mono-dechlorinated CP, CPO, mono-O-deethyl CP, and mono-O-deethyl CPO as well as the 6-mercapturic acid conjugate of 3,5-dichloro-2-pyridinol. The presence of 3,5-dichloro-6-methylthio-2-pyridinol as well as its O-glucuronide suggests further a cysteine S-conjugate beta-lyase mediated degradation. In addition to the qualitative LC-MS/MS screening the renal elimination profiles of the primary products of scheme (1), i.e. diethyl thiophosphate (DETP), diethyl phosphate (DEP), and 3,5,6-trichloro-2-pyridinol (TCP), were monitored over 14 days (n=21). A biphasic first-order excretion mechanism with half-lives of 21.5h (initial fast excretion phase) and 119.5h (terminal phase) for the sum of free DETP and DEP was found. TCP was hardly eliminated in its free form (O-glucuronide identified as phase II conjugate) and half-lives calculated for the total amount of TCP (acidic hydrolysis of urine samples) were 40.8 and 150.7h. The present study gives a more detailed view on the biotransformation of CP and together with the obtained kinetic data adds novel aspects to the limited knowledge of human metabolism of this xenobiotic, in particular at high dosage.