The effect of age on the rate of uptake (Jd) of a homologous series of saturated fatty acids into rabbit jejunum was determined in suckling, mature, and old animals. A previously validated in vitro technique was used, and the bulk phase was stirred at different rates to vary the effective resistance of the unstirred water layer (UWL). When UWL was high, Jd of fatty acids was greater in old than in young animals; Jd was greatly increased when UWL was low, and Jd was not higher in the suckling and mature rather than the old animals. At each rate of stirring UWL was lowest in the suckling rabbits and highest in the old animals. The values of Jd were corrected for UWL, yielding estimates of the true passive permeability properties of the membrane. From these values, the incremental free energy change associated with the addition of each -CH2- group to the fatty acid chain was 50% higher in the suckling and mature animals than in the old animals, and the estimated surface area of the jejunal membrane was also higher in the suckling and mature animals. Variations in UWL had a profound effect on the Jd of passively absorbed fatty acids, and failure to account for UWL leads to gross underestimation of the permeability properties of the jejunal microvillus membrane. Furthermore, the difference between the true and apparent passive permeability coefficients becomes greater as the fatty acid chain length increases. The results suggest that discrepancies in uptake of fatty acids in animals of different ages are explained by differences in the passive permeability properties and functional surface area of the membrane and by differences in the overlying UWL.