It is now generally accepted that dietary lipids permeate through the cholesterol-phospholipid bilayer of the intestinal microvillus membrane during the process of intestinal absorption. Therefore, it has been assumed that rates of lipid permeation depend upon the physical properties of the microvillus membrane. In this study the lipid permeability properties of the microvillus membrane were compared in two regions of the intestine, jejunum and ileum. Since the jejunum is exposed to the majority of dietary lipid it would be reasonable to suppose that it would be more efficient at lipid absorption. The ileum was found to be less permeable to all fatty acids examined and this could be correlated with increased rigidity of ileal microvillus membrane vesicles measured with multiple fluorescent probes. Differences in membrane fluidity were found in both the outer third and central regions of the bilayer. When measurements of membrane fluidity were performed either in the presence or the absence of fatty acids, it could be demonstrated that these acids perturb the physical properties of the outer region or the membrane. Therefore, this suggests that the rate-limiting step in fatty acid permeation may be localized to the outer third of the bilayer. Pharmacologic or dietary manipulations attempting to alter rates of lipid permeability should, therefore, be directed towards altering the physical properties of this region of the microvillus membrane.