Aims: To determine the effects of verapamil and diltiazem on simvastatin metabolism in human liver microsomes and to compare their inhibitory potencies and CYP3A4 inactivation parameters with those reported previously for mibefradil. Methods: Simvastatin metabolism was investigated in human liver microsomes in the presence and absence of verapamil or diltiazem (0.1-250 μM). Kinetics of CYP3A4 inactivation by verapamil and diltiazem were determined using testosterone as the substrate. Results: When verapamil was coincubated with simvastatin, IC50 values ranged from 23 to 26 μM for all major metabolites. The IC50 values ranged from 4.8 to 5.6 μM on preincubation of verapamil for 30 min in the presence of an NADPH-generating system. Corresponding IC50 values for diltiazem ranged from1 10-127 μM and from 21-27 μM, respectively. Verapamil and diltiazem inhibited testosterone 6β-hydroxylation in a time- and concentration-dependent manner, key features of mechanism-based inactivation. Values for the inactivation parameters kinact and KI were 0.15 ± 0.04 min-1 (mean ± s.d.) and 2.9 ± 0.6 μM, respectively, for verapamil and 0.07 ± 0.01 min-1 and 3.3 ± 1.5 μM, respectively, for diltiazem. Conclusions: The IC50 values for coincubation of verapamil and diltiazem were 46- and 220-fold higher, respectively, than those reported previously for mibefradil, and 16- and 71-fold higher, respectively, for preincubation. Thus, the results of this study suggest that verapamil and diltiazem are less likely than mibefradil to cause acute drug interactions with simvastatin in vivo. However, verapamil and diltiazem are moderate mechanism-based inhibitors of CYP3A4 and therefore may still cause significant inhibition of simvastatin metabolism in vivo during chronic therapy.