Studies on the intestinal transport of weak acids suggest that the subepithelial tissues exhibit a modest, but significant, ability to discriminate between the ionized and nonionized forms. This suggestion has been tested directly in experiments using an in vitro preparation of rat small intestine from which the epithelial cells were removed, but in which the structural and functional integrity of the subepithelial tissues was maintained. Studies on the effects of potential difference on the fluxes of weak acids in this preparation showed that the ratio of permeabilities for the ionized and nonionized species (Pi/Pni) was indeed less than one, and of a magnitude comparable to the value suggested by analysis of transport in the intact tissue. (Pi/Pni) for the subepithelial tissue decreased as pH was increased, and the discriminatory properties of the tissue were abolished [(Pi/Pni)=1] on treatment with the cationic macromolecule polyethyleneimine (PEI). These observations suggested that the discriminatory properties of the subepithelial tissues were determined by fixed anionic sites, and morphological studies with PEI indicated that such sites were concentrated in the region of the basement membrane.