The concurrent use of herbal medicinals with prescription and over-the-counter drugs carries a risk for unanticipated adverse drug-botanical pharmacokinetic interactions, particularly as a result of cytochrome P450 (P450) inhibition. Extracts of goldenseal (Hydrastis canadensis) containing approximately equal concentrations ( approximately 17 mM) of two methylenedioxyphenyl alkaloids, berberine and hydrastine, inhibited with increasing potency (CYP2C9) diclofenac 4'-hydroxylation, (CYP2D6) bufuralol 1'-hydroxylation, and (CYP3A4) testosterone 6beta-hydroxylation activities in human hepatic microsomes. The inhibition of testosterone 6beta-hydroxylation activity was noncompetitive with an apparent Ki of 0.11% extract. Of the methylenedioxyphenyl alkaloids, berberine (IC50 = 45 microM) was the more inhibitory toward bufuralol 1'-hydroxylation and hydrastine (IC50 approximately 350 microM for both isomers), toward diclofenac 4'-hydroxylation. For testosterone 6beta-hydroxylation, berberine was the least inhibitory component (IC50 approximately 400 microM). Hydrastine inhibited testosterone 6beta-hydroxylation with IC50 values for the (+)- and (-)-isomers of 25 and 30 microM, respectively. For (-)-hydrastine, an apparent Ki value of 18 microM without preincubation and an NADPH-dependent mechanism-based inhibition with a kinactivation of 0.23 min(-1) and a KI of approximately 110 microM were determined. Cytochrome P450 metabolic-intermediate (MI) complex formation could be demonstrated for both hydrastine isomers. With expressed P450 isoforms, hydrastine formed a P450 MI complex with CYP2C9, CYP2D6, and CYP3A4. Coexpression of cytochrome b5 with the P450 isoforms enhanced the rate but not the extent of P450 MI complex formation.