Fatty acids and cholesterol permeate across the intestinal microvillus membrane at rates dictated by the hydrophobicity of the permeating lipid and the permeability properties of the microvillus membrane. A theory has evolved suggesting that the chemical composition and physical properties of the microvillus membrane are important in determining microvillus membrane lipid permeability in vivo. This communication reports a test of this hypothesis. To compare in vivo membrane lipid permeability within the same intestinal region, but under conditions were membrane physical properties were radically altered, rats were fed an inhibitor of cholesterol synthesis. This resulted in the replacement of 87-90% of membrane cholesterol with its' precursor, 7-dehydrocholesterol. Marked changes in membrane physical properties were observed, including a reduction in the static and dynamic component of membrane fluidity within the jejunal microvillus membrane. These changes were limited primarily to the outer regions of the bilayer. Associated with these alterations was a pronounced reduction in membrane lipid permeability. Therefore, microvillus membrane lipid permeability, in vivo, appears to be correlated with physical properties of the bilayer, especially those of the superficial regions.