Airway surface liquid (ASL) pH has been proposed to be important in the pathophysiology of cystic fibrosis, asthma, and cough. Ratio image analysis was used to measure pH in the ASL after staining with the fluorescent pH indicator 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-dextran. ASL pH in bovine airway cell cultures grown at an air-liquid interface was 6.98 +/- 0.06 in the absence and 6.81 +/- 0.04 in the presence of HCO/CO(2). Steady-state ASL pH changed in parallel to changes in bath pH and was acidified by Na(+) or Cl(-) replacement but was not affected by the inhibitors amiloride, glibenclamide, or 4,4'-dinitrostilbene-2,2'-disulfonic acid. In response to sudden acidification or alkalization of the ASL by approximately 0.4 pH units by HCl/NaOH, ASL pH recovered to its initial value at a rate of 0.035 pH units/min (-HCO) and 0.060 pH units/min (+HCO); the pH recovery rate was reduced by amiloride and H(2)DIDS. In anesthetized mice in which the trachea was surgically exposed for measurement of BCECF-dextran fluorescence through the translucent tracheal wall, ASL pH was 7.14 +/- 0.01. ASL pH was sensitive to changes in blood pH created by metabolic (HCl or NaHCO(3) infusion) or respiratory (hyperventilation, hypoventilation) mechanisms. ASL pH is thus primarily determined by basolateral fluid pH, and H(+)/OH(-) transport between the ASL and basolateral fluid involves amiloride-sensitive Na(+)/H(+) exchange and stilbene-sensitive Cl(-)/HCO exchange. The rapid response of ASL pH to changes in systemic acid-base status may contribute to airway hypersensitivity in asthma and other airway diseases.