Silibinin, the main constituent of silymarin, a flavonoid drug from silybum marianum used in liver disease, was tested for inhibition of human cytochrome P-450 enzymes. Metabolic activities were determined in liver microsomes from two donors using selective substrates. With each substrate, incubations were carried out with and without silibinin (concentrations 3.7–300 μM) at 37° in 0.1 M KH2PO4 buffer containing up to 3% DMSO. Metabolite concentrations were determined by HPLC or direct spectroscopy. First, silibinin IC50 values were determined for each substrate at respective KM concentrations. Silibinin had little effect (IC50>200 μM) on the metabolism of erythromycin (CYP3A4), chlorzoxazone (CYP2E1), S(+)-mephenytoin (CYP2C19), caffeine (CYP1A2) or coumarin (CYP2A6). A moderate effect was observed for high affinity dextromethorphan metabolism (CYP2D6) in one of the microsomes samples tested only (IC50=173 μM). Clear inhibition was found for denitronifedipine oxidation (CYP3A4; IC50=29 μM and 46 μM) and S(−)-warfarin 7-hydroxylation (CYP2C9; IC50=43 μM and 45 μM). When additional substrate concentrations were tested to assess enzyme kinetics, silibinin was a potent competitive inhibitor of dextromethorphan metabolism at the low affinity site, which is not CYP2D6 (Ki,c=2.3 μM and 2.4 μM). Inhibition was competitive for S(−)-warfarin 7-hydroxylation (Ki,c=18 μM and 19 μM) and mainly non-competitive for denitronifedipine oxidation (Ki,n=9 μM and 12 μM). With therapeutic silibinin peak plasma concentrations of 0.6 μM and biliary concentrations up to 200 μM, metabolic interactions with xenobiotics metabolised by CYP3A4 or CYP2C9 cannot be excluded.