The purpose of this study was to compare the kinetics of intestinal and hepatic cytochrome P-450 3A (CYP3A) inhibition by using microsomal midazolam 1'-hydroxylation as a marker of enzyme activity. The effect of two antifungal agents commonly implicated in CYP3A drug-drug interactions was examined. Inhibition type and affinities were determined for human liver and intestinal microsomes screened for the presence or absence of CYP3A4 and CYP3A5, as well as for cDNA-expressed CYP3A4 and CYP3A5 microsomes. Ketoconazole and fluconazole were found to be noncompetitive inhibitors of both enzymes. Ketoconazole exhibited a Ki for cDNA-expressed CYP3A4 of 26. 7 +/- 1.71 nM, whereas the Ki for cDNA expressed CYP3A5 was 109 +/- 19.7 nM. Corresponding Ki values for fluconazole were 9.21 +/- 0.51 microM and 84.6 +/- 12.9 microM. For liver and intestinal microsomes that contained only CYP3A4, the average ketoconazole Ki was found to be 14.9 +/- 6.7 nM and 17.0 +/- 7.9 nM, respectively, whereas fluconazole yielded mean respective Ki values of 10.7 +/- 4.2 microM and 10.4 +/- 2.9 microM. Liver and intestinal microsomes that contained an equal or greater amount of CYP3A5, in addition to CYP3A4, were less susceptible to inhibition by both ketoconazole and fluconazole. These findings suggest that there can be significant differences in the affinity of these two enzymes for inhibitors. This may further broaden interindividual variability with respect to the magnitude of in vivo drug-drug interactions. We also conclude that there is no significant difference in inhibition type and affinity of ketoconazole and fluconazole for hepatic versus intestinal CYP3A4.