Intracellular pH (pHi) of the surface mucous cells of the rat stomach was measured in vivo using a fluorescent microscopic technique. Gastric surface mucous cells were loaded with fluorescein by superfusion with 5(6)-carboxy-fluorescein diacetate, and the ratio of the intensity of emitted fluorescence at 495 and 450 nm excitation was determined by image analysis of videotapes. An in vivo calibration curve was constructed using 10(-5) M nigericin to equilibrate pHi and extracellular pH, and intra- and extracellular potassium concentrations. The pHi of the gastric surface cells was 7.22 +/- 0.06, and pHi was stable over 45 min. A pulse of 20 mM NH4Cl in Krebs solution rapidly alkalinized the cells to pH 7.7, followed by a slow return of pHi to baseline pH 7.2. After withdrawal of NH4Cl, pHi dropped to 6.7 with gradual recovery to baseline. pHi increased when the mucosa was superfused, in the presence of nigericin, with 150 mM KCl solutions and decreased when superfused with 2.6 mM KCl solutions. Perfusing with pH 3 Krebs buffers transiently decreased pHi, whereas superfusion with pH 1.2 Krebs irreversibly decreased pHi. These findings confirm that the measured fluorescence was intracellular and that the cells responded to changes in luminal pH.