Transport characteristics of procainamide in the brush-border membrane isolated from rabbit small intestine were studied by a rapid-filtration technique. Procainamide uptake by brush-border membrane vesicles was stimulated by an outward H⁺ gradient (pH_in = 6.0, pH_out = 7.5) against a concentration gradient (overshoot phenomenon), and this stimulation was reduced when the H⁺ gradient was subjected to rapid dissipation by the presence of a protonophore, FCCP. An outward H⁺ gradient-dependent procainamide uptake was not caused by H⁺ diffusion potential. The initial uptake of procainamide was inhibited by other tertiary amines with N-dimethyl or N-diethyl moieties in their structures, such as triethylamine, dimethylaminoethyl chloride, and diphenhydramine, but not by tetraethylammonium and thiamine. Furthermore, procainamide uptake was stimulated by preloading the vesicles with these tertiary amines (trans-stimulation effect), indicating the existence of a specific transport system for tertiary amines. These findings indicate that procainamide transport in the intestinal brush-border membrane is mediated by the H⁺/tertiary amine antiport system that recognizes N-dimethyl or N-diethyl moieties in the structures of tertiary amines.