The effect of microsomal protein concentration on the inhibitory potency of a series of CYP3A inhibitors was assessed in vitro using diazepam 3-hydroxylation (yielding temazepam) as an index of CYP3A activity. With diazepam concentrations fixed at 100 micro M, inhibition of temazepam formation by fixed concentrations of ritonavir, ketoconazole, itraconazole, OH-itraconazole, norfluoxetine, and fluvoxamine decreased substantially as active protein concentrations increased from 0.0625 to 3.0 mg/ml. However protein concentration had only a small effect on the inhibitory activity of fluconazole. Equilibrium dialysis indicated extensive microsomal binding of all inhibitors except fluconazole; binding increased with higher protein concentrations. Based on the CYP3A content of liver microsomes, decrements in inhibitory potency of stronger inhibitors (ketoconazole and ritonavir) could be explained by specific binding, whereas nonspecific binding is anticipated to account for the effect on weaker inhibitors (norfluoxetine and fluvoxamine). Thus, microsomal binding (specific, nonspecific, or a combination of both) may have a major effect on estimation of inhibitory potency of p450 inhibitors and may contribute to variations among laboratories. The effect can be minimized by use of the lowest possible microsomal protein concentration for in vitro studies of metabolic inhibition.