The metabolism of tramadol was investigated in vitro using microsomal fractions of human liver. The parent compound and its main metabolites were determined by a newly developed high performance liquid chromatography assay. O-demethylation of tramadol was found to be stereoselective. The V_max of the O-demethylation of (–)-tramadol was 210 pmol·mg^–·min^–1, whereas (+)-tramadol was O-demethylated with a V_max of 125 pmol·mg^–1·min^–1. The K_m for both enantiomers was determined to be 210 M. O-demethylation was inhibited competitively by quinidine (k_i=15 nM) and propafenone (k_i=34 nM). N-demethylation was also stereoselective, preferentially metabolizing the (+)-enantiomer. Whereas O-demethylation displayed monophasic Michaelis-Menten kinetics, N-demethylation was best described by a two-site model. Competitive inhibition of the O-demethylation both by quinidine and propafenone suggests that O-demethylation is carried out by P-450IID6.