Aromatic hydroxylation has been established as a pathway for the in vivo metabolism of 3,4-(methylenedioxy)methamphetamine (MDMA) in the rat. Hydroxylation occurred at positions 2, 5 and 6 of the 3,4-methylenedioxyphenyl ring, but is favored at the 6 position. All three regioisomers of both hydroxy-MDMA and hydroxy-3,4-(methylenedioxy)amphetamine (hydroxy-MDA) were detected in the rat liver when 20 mg kg-1 of MDMA was administered. However, 6-hydroxy-MDMA and 6-hydroxy-MDA were the only hydroxylated metabolites detected in the rat brain and plasma and no hydroxylated metabolites were detected in the urine. The hydroxylated metabolites were identified by co-injection of synthetic reference compounds and comparison of the mass spectra of the trifluoroacetyl derivatives of the metabolites with the synthesized reference compounds. The regioisomers of both hydroxy-MDMA and hydroxy-MDA could not be distinguished by either single-stage or two-stage mass analysis. However, employment of a third stage of mass analysis produced distinctly different mass spectra for each of the three regioisomers.