The role of the preepithelial mucus-HCO-3 layer in protection against intracellular acidosis was investigated in isolated Necturus gastric antral mucosa exposed to luminal acid by simultaneous measurement of intracellular pH (pH(i)) and extracellular surface pH (pHs) in surface epithelium with microelectrode technique. Acidification of the luminal perfusate to pH 2.5 acidified pH(i) in surface epithelial cells from 7.33 +/- 0.02 to 7.20 +/- 0.04, whereas pHs fell from 6.75 +/- 0.21 to 5.20 +/- 0.25 (P < 0.01; n = 9), followed by a steady state for at least 2 h. Inhibition of epithelial HCO-3 secretion and transport by removal of serosal HCO-3 and CO2 (HEPES and O2 substitution) during acid exposure provoked a progressive acidification of pHs from 5.60 +/- 0.41 to 2.74 +/- 0.14 in 30 min (P < 0.01; n = 9), which was accompanied, after a 5- to 10-min delay, by acidification of pH(i) from 7.21 +/- 0.03 to 5.68 +/- 0.26 (P < 0.01). Digestion of the surface mucus gel by pepsin (5% wt/vol) at pH 2.5 caused a slow acidification of pHs from 5.22 +/- 0.59 to 3.60 +/- 0.46 within 2 h. This was followed by a more rapid acidification to 2.53 +/- 0.38 (P < 0.01; n = 7), with concomitant acidification of pH(i) from 7.19 +/- 0.05 to 6.03 +/- 0.33 (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)