The influence of enzyme localization and blood flow on intestinal elimination was evaluated in rats. Phenol was administered vascularly (∼1400 and 2500 μg) and luminally (intrajejunal bolus doses of ∼100 and 1000 μg) to the recirculating in situ perfused intestine. The portal effluent and the reservoir were sampled. The intestinal extraction ratios for phenol at the low and high vascular doses were (mean ± S.D., n = 3) 0.09 ± 0.02 and 0.11 ± 0.01, respectively. The perfusion flow rate was also varied from 5 to 12 ml/min at a vascular dose of ∼2500 μg of phenol. The organ clearance at the lowest flow rate significantly exceeded those at the higher flow rates. The presence of a diffusional barrier at the mucosa-serosa interface was suggested. The calculated mean diffusional clearance of phenol was 1.11 ml/min. Sulfation was the predominant metabolic pathway after vascular administration of phenol. After luminal dosing, the intestinal intrinsic clearances of phenol at the low and high doses were 7.29 ± 1.39 (n = 4) and 3.55 ± 1.16 ml/min (n = 3), respectively, indicating saturation at the higher dose. Moreover, there was a decrease in the area under the curve ratio (metabolite/phenol) at the high luminal dose. Luminal administration, in general, produced greater glucuronidation. These data and STELLA simulations suggest that enzyme localization at both the cellular and tissue levels has a significant influence on intestinal metabolism.