Oxybutynin has an extensive first pass metabolism after oral administration, the main active metabolite being N-desethyloxybutynin. The purpose of this study was to investigate the CYP isoform specificity of oxybutynin N-deethylation and possible interactions. Oxybutynin N-deethylation in human liver microsomes in vitro was potently inhibited by ketoconazole (IC50 4.5 μM), less and variably by itraconazole and not by quinidine or several other reference inhibitors, suggesting that CYP3A enzymes are predominant catalysts of the reaction. Recombinant CYP3A5 enzyme had higher activity in oxybutynin N-deethylation than recombinant CYP3A4. Ketoconazole inhibited oxybutynin N-deethylation by the recombinant CYP3A4 and CYP3A5 almost completely, whereas itraconazole inhibited the activity of CYP3A4 more potently than that of CYP3A5. Oxybutynin inhibited CYP3A4- and CYP2D6- associated activities (testosterone 6β-hydroxylase and dextromethorphan O- demethylase, respectively) in human liver microsomes. CYP1A1/2-, CYP2A6-, CYP2C9- and CYP2E1-associated activities were inhibited less potently or not at all by oxybutynin when compared with reference inhibitors. Although the reasons for the weak and variable inhibition by itraconazole remain to be studied, it seems that oxybutynin is predominantly metabolized by CYP3A4 and CYP3A5 but not by CYP2D6. However, it seems to have some affinity also to the latter enzyme.