1. Metabolism of the analgesic agent tramadol hydrochloride has been investigated after a single oral administration of tramadol to three male volunteers (100 mg/subject), and a urine pool (4-12h) was obtained. 2. Unchanged tramadol and a total of 23 metabolites, consisting of 11 Phase I metabolites (M1-11) and 12 conjugates (seven glucuronides, five sulphates), were profiled, characterized and tentatively identified in urine on the basis of API ionspray-MS and MS/MS data. 3. Of the metabolites, five (M1-5) had been previously identified. 4. The metabolites were formed via the following six metabolic pathways: (1) O-demethylation, (2) N-demethylation, (3) cyclohexyl oxidation, (4) oxidative N-dealkylation, (5) dehydration and (6) conjugation. 5. Pathways 1-3 appear to be major routes, forming seven O-desmethyl/N-desmethyl and hydroxycyclohexyl metabolites. 6. Pathways 1-3 in conjunction with pathway 6 produced seven glucuronides along with five sulphates. 7. In addition, the in vitro metabolism of tramadol was conducted using a human liver microsomal fraction in the presence of an NADPH-generating system. Unchanged tramadol (82% of the sample) plus eight metabolites (M1, M2, M4-6, tramadol-N-oxide (M31), OH-cyclohexyl-M1 (M32) and dehydrated tramadol-N-oxide), were profiled and tentatively identified on the basis of MS and MS/MS data.