1. The in vivo biotransformation of (+/-)-3,4-methylenedioxymethamphetamine [(+/-)-MDMA] in the thoroughbred horse was determined after oral administration. 2. Unconjugated compounds and aglycones were isolated from enzyme-hydrolysed urine by solid-phase extraction using mixed-mode cartridges. The basic isolates were derivatized (trimethylsilylether, TMS) and analysed by positive-ion electron ionization/gas chromatography-mass spectrometry (EI+/GC-MS). MDMA and 10 Phase I metabolites containing the arylisopropylamine substructure were detected. 3. N-Hydroxy amphetamine and N-hydroxymethamphetamine were synthesized. The EI + mass spectra of their O-TMS derivatives showed characteristic alpha-cleavage ions at m/z 132 and 146, respectively, as base peaks. Based upon these data, five putative N-hydroxylated metabolites of MDMA were detected. 4. In the horse, (+/-)-MDMA is metabolized by oxidative N-demethylation to form the primary amine methylenedioxyamphetamine (MDA). Both MDMA and MDA are further metabolized by oxidative demethylenation (cleavage and O-demethylation of the benzodioxole moiety) to form the corresponding catechols, 3-O-methylation to form the guaiacols and N-oxidation of the secondary and primary amine metabolites to form the hydroxylamines. 5. Both phenolic and N-hydroxy metabolites of (+/-)-MDMA undergo Phase II conjugation before excretion in urine.