Dextromethorphan (DMO), a cough suppressing synthetic analog of codeine, undergoes parallel O-demethylation to dextrorphan (DOP), and N-demethylation to 3-methoxymorphinan (MEM), in humans. 3-hydroxymorphinan, a didemethylated metabolite, is formed secondarily. O-demethylation activity is well established as an index reaction for CYP2D6. However, this pathway appears to be mediated by at least two different enzymes in vitro. N-demethylation activity has recently been proposed to reflect CYP3A3/4 activity. We investigated both pathways in vitro with microsomal preparations from three human livers to assess the value of DMO as a probe drug for CYP2D6 and CYP3A3/4, DMO O-demethylation displayed a biphasic pattern with a high-affinity site reflecting CYP2D6 activity (mean Ki for quinidine, 0.1 +/- 0.13 microM). Kinetic parameters for the two O-demethylation mediating enzymes predict an average relative intrinsic clearance (Vmax/K(m) ratio) of 96% of total O-demethylation mediated via the high-affinity enzyme. Thus, in vitro data confirms the usefulness of DMO O-demethylation as an index reaction to monitor CYP2D6 activity. The Eadie-Hofstee plot of DMO N-demethylation was consistent with single-enzyme Michaelis-Menten kinetics (Vmax varying from 3.3 to 6.8 nmol mg-1 min-1, K(m) from 231 to 322 microM). However, ketoconazole, a CYP3A3/4 inhibitor, reduced N-demethylation only by 60% and had a mean Ki an order of magnitude higher (0.37 microM) compared to other pure CYP3A3/4 mediated reactions. Troleandomycin, a mechanism based CYP3A3/4 inhibitor, inhibited MEM formation by an average maximum of 46%, with an IC50 varying from 1 to 2.6 microM. A polyclonal rat liver CYP3A1 antibody inhibited MEM formation only by approximately 50%. Diethyldithiocarbamate (DDC), a mechanism based CYP2E1 inhibitor, reduced MEM formation at concentrations up to 150 microM between 33 and 43%. Chemical inhibitors of CYP2d6 (quinidine), CYP1A1/2 (alpha-naphthoflavone), and CYP2C9 (sulfaphenazole), as well as a goat rat liver CYP2C11 polyclonal antibody (inhibitory against human CYP2C9 and CYP2C19), had minimal effect on MEM formation rate, thus excluding an involvement of any of these enzymes. DMO N-demethylation is only partly mediated by CYP3A3/4, and therefore is not a reliable index reaction for CYP3A3/4 activity either in vitro or probably in vivo.