Intracellular pH (pHi) and viability of gastric surface cells of the rat stomach in response to luminal acidification, and the role of Na+/H+ exchange in maintaining pHi homeostasis were studied in vivo using a fluorescent microscopic technique. pHi was measured during superfusion with buffers of pH 1.2-7.4. When the pH of the superfusate was 7.4, baseline pHi was unchanged. Superfusion with pH 3 buffer rapidly decreased pHi to 6.7, with subsequent recovery to baseline pHi within 15 min despite continuing acid exposure. Superfusion with buffers of pH 1.7 and 1.2 decreased pHi continuously to below 6.2 with no recovery observed. Despite the relentless decline in pHi during superfusion with pH-1.2 and -1.7 solutions, over 75% of the surface cells were still viable, as measured by exclusion of the vital dye propidium iodide. We then examined the role of Na+/H+ exchange in the regulation of pHi. Superfusion with amiloride did not affect recovery of pHi from intracellular acidification induced by a NH4Cl prepulse. Exposure to the potent, lipophilic Na+/H+ exchange inhibitor 5-(N,N-hexamethylene)-amiloride (HMA), either in the superfusate or by close arterial perfusion, decreased baseline pHi from 7.1 to 6.8. Close arterial perfusion of HMA additionally attenuated the recovery of pHi to baseline during superfusion with pH 3 buffer. We conclude that luminal protons permeate into the cytoplasm of gastric surface cells, where they are eliminated by an Na+/H+ exchanger, most probably localized to the basolateral membrane.