Passive permeation into the intestine is influenced by resistance of the unstirred water layer (UWL) and the microvillus membrane. Failure to account for UWL leads to gross underestimation of apparent passive permeability coefficients, and to qualitative errors in the interpretation of the nature of absorptive processes. A previously validated in vitro technique was used 1) to determine the permeability characteristics of the rabbit jejunum (J) and ileum (I) towards a homologous series of saturated fatty acids; 2) to measure UWL in jejunum and ileum under conditions of variable effective resistance of the UWL; and 3) to estimate the preferential site of absorption of cholesterol in the intestine. The rate of uptake, Jd, of fatty acids was similar in J and I when UWL was high; although Jd was higher in J than I when UWL was low, the UWL was smaller in I than J at each rate of stirring of the bulk phase, and the incremental free energy change for the uptake of medium chain-length fatty acids was similar in J and I, indicating similar passive permeability characteristics along the length of the intestine. However, the intercept of the relationship between chain length and uptake was significantly different for the two sites, suggesting a greater surface area, Sm, of the membrane in J than I. These findings persisted over a wide range of different periods of incubation and of concentrations of cholesterol and bile acids. The results suggest that i) cholesterol uptake in the J and I occurs from an aqueous monomolecular phase into the microvillus membrane; and ii) reported variations in the Jd of cholesterol along the intestine are likely due to differences in UWL, Sm, or availability of substrate and not to differences in the permeability properties of the intestine.