The utility of microdialysis as a quantitative sampling technique for in vitro drug metabolism studies was demonstrated by investigating the stereoselective metabolism of d-, l- and dl-amphetamine by the cytochrome P-450 enzymes. Microdialysates containing the isomers of amphetamine and its metabolite were derivatized with the fluorescent chiral derivatizing agent, (-)-fluorenylethyl chloroformate. The diastereoisomers were isocratically separated by liquid chromatography (LC) on a reversed-phase C 1 8 , 3-μm (100x3.2 mm) column. The intra- and inter-assay relative standard deviation (R.S.D.) was below 10%. Michaelis-Menten parameters, K m and V m a x were obtained for the formation of both d- and l-hydroxyamphetamine from d-, l- and dl-amphetamine in the concentration range of 10-350 μM.