We measured the pH of airway surface liquid (ASL) secreted by the ferret trachea in vitro by using a catheter-tipped pH electrode implanted in a collecting cannula close to the airway epithelium. Mucus secretion was promoted by methacholine (0.02 mmol/l) in the organ bath. The pH of the ASL was 6.85 +/- 0.03 (SE) compared with a bath value of 7.39 +/- 0.01, when the bath was bubbled with 5.65% CO2. Changing the bath CO2 from 0 to 20.93% CO2 altered the bath pH from 8.06 to 6.96, but the ASL pH only varied from 6.92 to 6.85. This homeostasis of ASL pH was not the result of the buffering powers of the ASL, because ex situ buffer curves for secreted ASL were similar to those for Krebs-Henseleit solution. Changing the luminal CO2 content by blowing gases through the trachea changed ASL pH by values similar to that ex situ. However, when external organ bath CO2 was changed, the luminal CO2 changes were proportionately far smaller. Measurement of rates of diffusion of CO2 across the tracheal wall indicated that this was not a limiting factor in the results. Similarly, measurement of metabolic rate CO2 production in the tracheal lumen indicated that this did not significantly affect the results. We conclude that the pH of ASL is significantly on the acid side of the pH or interstitial fluid and plasma and that it is maintained relatively constant despite large changes in external pH.