Intracellular pH (pHi) was measured using the fluorescent dye 2'7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) in cells of isolated rabbit gastric glands that were either suspended in a stirred cuvet or attached to a cover slip. Glands were treated with HCO3-free Ringer's (100% O2 gassing) of different ionic composition and extracellular pH (pHo). In NaCl Ringer's steady state pHi = 7.15 was independent of pHo over the range pHo 7.1-7.8. This regulatory range of pHi (= 0.7 pH units) was abolished by sodium-free Ringer's or addition of 10(-3) M amiloride and also by 10(-4) M ouabain. Chloride-free Ringer's or 2 X 10(-4) M SITS caused an elevation of pHi to 7.5, and the regulatory range was reduced in magnitude to 0.50 pH units (pHo 7.3-7.8). At pHo values outside the regulatory ranges, pHi was linearly related to pHo, although the specific slope that related pHi to pHo depended on the composition of the external solution. In potassium gluconate Ringer's, pHi was completely independent of pHo. The data suggest that Na/H exchange is primarily responsible for the regulation of pHi over a wide range of pHo, although the specific pHi and the range of regulation are affected by the activity of the Cl/OH (HCO3) exchanger; the movement of protons or hydroxyl ions, or both, across gland cell membranes appears to be mediated under most circumstances by these two exchangers.