Cytochrome P450 3A (CYP3A) metabolizes a diverse array of clinically important drugs. For some of these (e.g., cyclosporine, verapamil, midazolam), CYP3A in the intestinal mucosa contributes to their extensive and variable first-pass extraction. To further characterize this phenomenon, we measured CYP3A content and catalytic activity toward the probe substrate midazolam in mucosa isolated from duodenal, jejunal and ileal sections of 20 human donor intestines. For comparison, the same measurements were performed for 20 human donor livers, eight of which were obtained from the same donors as eight of the intestines. Excellent correlations existed between homogenate and microsomal CYP3A content for the three intestinal regions. Median microsomal CYP3A content was greatest in the duodenum and lowest in the ileum (31 vs. 17 pmol/mg of protein). With respect to midazolam 1'-hydroxylation kinetics, the median Km for each intestinal region was similar to the median hepatic Km, approximately 4 microM. In contrast, the median Vmax decreased from liver to duodenum to jejunum to ileum (850 vs. 644 vs. 426 vs. 68 pmol/min/mg). Intrinsic clearance (Vmax/Km) followed a similar trend for the intestinal regions; median duodenal intrinsic clearance was comparable to hepatic intrinsic clearance (157 and 200 microl/min/mg, respectively). Vmax correlated with CYP3A content for all tissues except the ileum. Duodenal and jejunal Vmax and CYP3A content varied by >30-fold among donors. Microsomes prepared from every other 1-foot section of six intestines were also analyzed for CYP3A as well as for two coenzymes. In general, CYP3A activity, CYP3A content and CYP reductase activity rose slightly from duodenum to middle jejunum and then declined to distal jejunum and ileum. Cytochrome b5 content and cytochrome b5 reductase activity varied little throughout the intestinal tract. Regional intrinsic midazolam 1'-hydroxylation clearance was greatest for the jejunum, followed by the duodenum and ileum (144, 50 and 19 ml/min, respectively). Collectively, these results demonstrate that the upper small intestine serves as the major site for intestinal CYP3A-mediated first-pass metabolism and provides a rationale for interindividual differences in oral bioavailability for some CYP3A substrates.