The retinal pigment epithelium (RPE) interposed between the vascular system of the choroid and the neural retina performs a variety of functions essential for vision. In order to further elucidate the transport functions of the RPE, apical membranes were isolated from the RPE of the dogfish (Squalus acanthias) by differential precipitation with calcium. Na-K-ATPase, an apical marker enzyme in this tissue, was enriched 15-fold in the final membrane fraction. About 50% of the membranes form right-side-out vesicles in which the membrane has retained its in vivo orientation. Sodium uptake into these vesicles as determined by a rapid filtration method was stimulated 37% by the presence of a proton gradient across the membrane (pHi = 6.1, pHo = 8.1). The stimulation was also observed in membrane vesicles "short-circuited" with valinomycin and K. The pH gradient-dependent sodium uptake but not the uptake in the absence of a pH gradient was completely inhibited by 5 X 10(-4) M amiloride, and 56% inhibition was found at 10(-5) M amiloride. The uptake of 22Na was also strongly decreased in the presence of nonradioactively labelled sodium and lithium; potassium was without effect. pH gradient dependence, amiloride sensitivity, saturability and cation specificity of the sodium flux indicate the presence of a Na/H exchanger in the apical membrane of the retinal pigment epithelium. The presence of the Na/H exchange process might have important implications for the control of pH in the subretinal space, optimum intracellular pH of the RPE and the triggering of other functions of the RPE.