Although cell culture models are increasingly used to study drug transport and metabolism, the influence of the substratum on the transport properties of the cell monolayer has not been studied in great detail. Furthermore, the use of effective (or apparent) permeabilities (Peff) assumes that the contribution of the microporous filter substratum and the aqueous boundary layer (ABL) to transport are negligible or are at least constant for a series of drugs. In the present study, the permeabilities of the substratum, ABL, and monolayer were obtained for a series of compounds at variable flow rates in side-by-side diffusion chambers. Comparisons of transport properties were made between cell monolayers grown on substrata made of polycarbonate (PC) and polyester (PE). All paracellular markers demonstrated a reduction in permeability and a corresponding increase in transepithelial electrical resistance (TEER) through PE-grown monolayers. The permeabilities of two carrier-mediated compounds, phenylalanine and proline, were 55% higher and 48% lower through PE-grown monolayers than through the PC-grown monolayers, respectively. The resistance to progesterone transport attributed to the PE and PC filters was large (71% and 27% of total resistance, respectively) at a flow rate of 20 mL/min, indicating that the monolayer was not the rate-limiting transport barrier. Therefore, for highly permeable compounds, reporting Peff has limited value since it is an indicator of the transport properties of the substratum rather than of the monolayer. These results demonstrate that substratum properties (e.g., membrane composition, pore size, etc.) significantly affect the barrier properties of the Caco-2 cell monolayer. The most probable mechanism is by the modulation of the functional expression of nutrient and ion transporters resulting in variable transcellular and paracellular transport properties. These results further demonstrate the importance of calculating intrinsic membrane transport parameters if the monolayer is not maintained as the rate-determining barrier in the transport experiment. Using higher flow rates and higher porosity substrata supports may help maintain the monolayer as the rate-limiting transport barrier.