1. The use of everted sacs of the small intestine as an enzyme source for the study of the first-pass metabolism of xenobiotics by cytochrome P450s (P450, CYP) is described. Several drug oxidation activities for testosterone, chlorzoxazone, tolbutamide, bufuralol and warfarin were observed when everted sacs (1-cm segment) from different parts of mouse small intestine were incubated with an NADPH-generating system and each substrate. 2. Most of the drug hydroxylase activities resided in the upper part of mouse small intestine and these activities were much higher than those of intestinal microsomes. Drug oxidation activities decreased along the distance from the upper part of the small intestine except for warfarin hydroxylation. 3. Testosterone 6beta-hydroxylation in the everted sacs exhibited the highest catalytic activities among the drug oxidations tested here. In the upper part of the small intestine, the testosterone 6beta-hydroxylase activities of everted sacs subjected once to freezing and thawing were substantially decreased compared with the untreated everted sacs. 4. Testosterone 6beta-hydroxylase activities in the everted sacs of the small intestine were significantly inhibited by ketoconazole. Immunoreactive proteins using anti-CYP3A antibodies were detected in the upper and middle parts of the small intestine. 5. The results demonstrated that the upper part of the mouse small intestine serves as the major site for intestinal P450 mediated first-pass metabolism. Everted sacs of the small intestine are therefore useful for the study of drug metabolism as well as of transport and absorption.