We investigated the effects of various phenothiazines on the Na+-H+ exchanger in brush-border membrane vesicles isolated from the proximal small intestine of the rabbit. The Na+-H+ exchanger activity was assayed by measuring the dimethylamiloride-sensitive Na+ uptake into these vesicles in the presence of an outward-directed H+ gradient ([pH](o) > [pH](i)). All phenothiazines that were tested inhibited the Na+-H+ exchanger and chlorpromazine sulfoxide (CPZ-SO) was the most potent inhibitor among them. CPZ-SO inhibited the dimethylamiloride-sensitive Na+ uptake in a dose-dependent manner and IC50 for this process was 90 μM. Even though CPZ-SO drastically reduced the initial rates of Na+ uptake, the equilibrium uptake of Na+ into the vesicles was not affected, indicating that the integrity of membrane vesicles was not altered by the drug. Furthermore, the inhibition of the exchanger activity by phenothiazines was not due to nonspecific dissipation of the H+ gradient, because CPZ-SO inhibited the dimethylamiloride-sensitive Na+ uptake even in the absence of a H+ gradient. The inhibition of the exchanger by CPZ-SO was freely and completely reversible. Kinetic analyses of the Na+-H+ exchanger carried out in the presence and absence of CPZ-SO revealed that the nature of the inhibition depended on the composition of the buffers used in the assay of the exchanger because the inhibition was of a mixed type in the mannitol medium, whereas it was of a competitive type in the KCl medium.