Two preparations of the guinea pig trachea have been examined: an isolated preparation and a preparation in vivo, both exposed to air on the mucosal surface. Ion-selective microelectrodes have been used to measure Na (alpha Na) and K activities (alpha K) in airway surface liquid (ASL) while airflows tending either to evaporate or to condense water were applied. Other variables measured included ASL depth and transepithelial potential difference (TEPD). In isolated preparations, condensation did not progressively alter depth, alpha Na, or TEPD but caused a slight increase in alpha K. Evaporation decreased depth; increased alpha Na, alpha K, and osmotic pressure; and changed TEPD. Measurements on preparations in vivo broadly supported these observations. In addition, the depth of ASL developed on isolated preparations was related to the humidity of the air to which animals had been previously exposed. We conclude that condensation and evaporation at the ASL-air interface in isolated preparations and in preparations in vivo do significantly modify key ASL variables as does the relative humidity of the air to which animals are exposed before experimentation.