Airway surface liquid (ASL) is a complex fluid with solutes including electrolytes, lipids, mucins, and proteins. The proximal airways are absorptive for most solutes, including proteins. We investigated the process of protein movement across confluent primary cultures of guinea pig trachea grown on filters using fluorescent-labeled bovine serum albumin (BSA), ovalbumin (OA), and 70-kDa dextran (Dex). We found marked asymmetry of BSA and OA transepithelial flux, with apical-to-basolateral flux (JA-->B) 10 times greater than the opposite direction (JB-->A) for both proteins. The apparent permeability for Dex was the same as that for proteins in the basolateral-to-apical direction and showed no asymmetry. Increasing concentrations of unlabeled BSA, OA, or transferrin inhibited JA-->B for both BSA and OA without affecting Dex movement. Cooling reduced JA-->B for BSA without affecting JB-->A. Monensin and nocodazole each reduced JA-->B for BSA and OA without affecting JB-->A. Monensin eliminated all asymmetry for BSA movement. Brefeldin A did not affect JA-->B for either protein but did increase JB-->A for BSA. Treatment with the protease inhibitors increased JA-->B for BSA. Western immunoblotting demonstrated immunologically intact protein in the downstream compartment. We conclude that there is transcytosis of proteins across cultured trachea epithelium in the apical-to-basolateral direction, which is monensin sensitive, involves microtubules, is not dependent on proteolysis, and is not protein species specific. This process may be important for maintenance of the ASL, and defects in this process may contribute to the abnormally thickened airway secretion seen in airway diseases.