The effect of carbonyl cyanide-m-chlorophenylhydrazone (CCCP) on Cl uptake across the brush-border membrane (BBM) was quantified using ³⁶Cl and BBM vesicles from guinea pig ileum. CCCP inhibited only partially both the pH gradient-activated Cl uptake and Cl/Cl exchange activities present in these vesicles. In contrast, CCCP had no effect on the initial (2-30 s) decay rate of an imposed proton gradient, as determined using the pH-sensitive fluorophore pyranine. Taken together, these results strongly indicate that the main action of CCCP does not consist of dissipating any imposed pH gradient but rather in inhibiting directly the pH gradient-activated Cl uptake and Cl/Cl exchange activities characterizing the intestinal BBM. Because these two activities can be explained in terms of a single (homogeneous) random, nonobligatory two-site Cl-H⁺ symporter, in which Cl/Cl exchange occurs by counterflow [F. Alvarado and M. Vasseur. Am. J. Physiol. 271 (Cell Physiol. 40): C1612-C1628, 1996], we developed a new, more general three-site symport model that fully explains the Cl uptake inhibitions caused by CCCP. This new model postulates the existence of a third, allosteric, inhibitory CCCP-binding site separate from either of the two substrate-binding sites of the Cl-H⁺ symporter, the Cl-binding and the H⁺-binding sites. Finally, we show that, to explain the partial inhibitions observed, it is necessary to postulate that all the substrate-bound carrier complexes, =C-S, I=C-S, A=C-S, and IA=C-S, where C is carrier, I is inhibitor, S is substrate, and A is activator, can form and be translocated.