The gastric (K, H)-ATPase has been shown to catalyze an electroneutral H+ for K+ exchange. Tl+ is able to substitute for K+ as an activating cation in the hydrolytic reaction with an apparent dissociation constant of 90 microM as compared to about 870 microM for K+. The ability of Tl+ to participate in transport is shown by the development of pH gradients in the presence of Tl+ following addition of ATP to gastric vesicles and by the ATP-dependent efflux of Tl+ from gastric vesicles. Inhibition of hydrolysis is observed at pH 7.4 with external Tl+ concentrations above 3.0 mM. This inhibition of hydrolysis is correlated with inhibition of pH-gradient formation. The inhibition of transport activity is partially relieved by a decrease in medium pH. This inhibitory effect is attributed to Tl+ binding at an external, low affinity cation site. In contrast to rubidium chloride, at high Tl+ concentrations, following the initial Tl+ efflux, there is reuptake of the cation. This rapid uptake is attributed to lipid-dependent Tl+ entry pathways. The vesicles exhibit a high permeability to thallium nitrate demonstrating a half-time (t1/2) for uptake of about 1.0 min in contrast to 46 min for rubidium chloride. In both gastric vesicles or liposomes, external Tl+ concentrations in excess of 1 to 4 mM are able to dissipate intravesicular proton gradients. Thus, although Tl+ is able to activate the gastric ATPase by mimicking K+, the permeability of this cation in lipid bilayers tends to uncouple H+ transport at concentrations high enough to generate detectable proton gradients.