Following oral administration of benztropine (IO mg/kg, body weight), the phase I metabolites, benztropine N-oxide, N-desmethylbenztropine, tropine, 4'-hydroxybenz- tropine, N-desmethyl-4'-hydroxybenztropine, 4'-hydroxvbenztropine N-oxide and methoxy-4'-hydroxybenztropine, together with unmetabolized benztropine, were isolated and identified in rat urine and bile by GC-electron impact mass spectrometry (EI GC/MS), microcolumn LC-electrospray mass spectrometry (ES LC/MS) and hplc followed by MS analysis. The mass spectra and chromatographic properties of isolated N-desmethylbenztropine, benztropine N-oxide and tropine were confirmed by comparison with authentic reference standards. Sufficient quantities of 4'-hydroxybenztropine and N-desmethyl-4'-hydroxybenztropine were isolated from the urine by tlc and examined by 1H-nmr, ES/MS and EI/MS. The structure of the methoxy-4'-hydroxybenztropine metabolite was determined by EI/MS. 4'-Hydroxybenztropine N-oxide was identified by reacting it with a reducing agent, titanous chloride, to form 4'-hydroxybenztropine, which was then confirmed by comparing its EI/MS and ES/MS behaviour with a previously isolated and 1H-nmr-authenticated sample. In addition, four intact glucuronide conjugates of benztropine were also characterized in bile and urine as phase II metabolites, including 4'-O-glucuronylbenzotropine, N-desmethyl-4'-O-glucuronylbenztropine, methoxy-4'-O-glucuronylbenztropine and 4'- O-glucuronylbenztropine N-oxide by hplc followed by ES/MS analysis. These results provide the first direct evidence of the presence of these metabolites of benztropine in rat.