Regulation of intracellular pH in gastric epithelial surface cells exposed to luminal acid was investigated in isolated Necturus antral mucosa using microelectrode technique. Exposure of the mucosa to luminal pH 2 acidified intracellular pH from 7.21 +/- 0.01 to 6.95 +/- 0.04 (N = 50). Removal of Na+ from the perfusates or addition of amiloride (1 mM) to serosal perfusate (containing HCO3-) had no influence on intracellular pH during exposure to pH 2 (N = 6), but removal of HCO3-/CO2 from or addition of 4, acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (0.5 mM) to the serosal perfusate (containing Na+) acidified intracellular pH from 7.02 +/- 0.03 to 6.45 +/- 0.15 (p less than 0.01, N = 10) and from 6.97 +/- 0.06 to 6.58 +/- 0.26 (p less than 0.01, N = 6), respectively, in 15 min. In tissues exposed to mucosal pH 6, epithelial surface pH was about 1.3 pH units higher than pH of the mucosal bulk solution. Removal of Cl-/HCO3- from the serosal perfusate acidified epithelial surface pH by about 0.5 pH units (p less than 0.01, N = 6), suggesting that serosal HCO3- sustains intracellular pH, at least in part, by generating an alkaline buffer layer at the epithelial surface. In the absence of HCO3-/CO2, a stable intracellular pH was obtained when the tissue was exposed to mucosal pH 2.7, but in this situation intracellular pH was sensitive to Na+ removal or amiloride addition, intracellular pH decreasing from 7.00 +/- 0.07 to 6.48 +/- 0.10 (p less than 0.01, N = 6) and from 6.86 +/- 0.06 to 6.32 +/- 0.01 (p less than 0.01, N = 7), respectively, in 15 min. The data suggest that in gastric epithelium exposed to luminal acid, physiological intracellular pH is primarily maintained by the buffer action of serosal HCO3- transported to the epithelial surface to impede the entry of luminal H+ into mucosal tissue. Removal of the sheltering HCO3- unmasks a second line, Na(+)-dependent and amiloride-sensitive intracellular pH regulatory mechanism, presumably a Na+/H+ antiport.