The aim of this study was to evaluate the effects of docosahexaenoic acid (DHA) on the intestinal cytochrome P450 isoenzyme (CYP3A) and P-glycoprotein (P-gp) functions using midazolam and rhodamine-123 as specific substrates of CYP3A and P-gp, respectively. Perfused everted intestinal segments from rats were employed to determine the effects of DHA on midazolam metabolism and rhodamine-123 transport. In addition, the effects of DHA on in vitro midazolam metabolism in rat intestinal microsomes and on midazolam bioavailability in rats were examined. The intestinal extraction ratio (ER G) of midazolam was determined to be 0.43 and decreased significantly to 0.12, 0.07, and 0.06 in the presence of 50, 100, and 200 microM DHA, respectively, in a concentration-dependent manner. The results from an in vitro study using rat intestinal microsomes demonstrated that DHA competitively inhibited the intestinal CYP3A activity with Ki of 15.7 and 27.1 microM for the formations of 1'-OH midazolam and 4-OH midazolam, respectively. Moreover, the oral administration of DHA (100mg/kg) increased the AUC infinity, Cmax, and oral bioavailability (F) of midazolam by about 50% in rats, without affecting the T 1/2, V dss/F, or CL tot/F. In contrast, DHA did not change the serosal-to-mucosal transport of rhodamine-123 in the perfused everted intestine and oral administration of DHA (100mg/kg) had no influence on the pharmacokinetics of intravenously administered midazolam in rats, thus suggesting that DHA has little effect on the intestinal P-gp activity and hepatic clearance of midazolam. This study provided the first direct evidence to show that DHA has an inhibitory effect on the intestinal pre-systemic metabolism of a CYP3A substrate and that DHA has little, if any, effect on the P-gp activity in the gut.