The mucosal surface of the conducting airways has specialized structures for respiratory defense. Glands secret mucus that may act as a barrier to particle penetration and participate in particle clearance. Intraepithelial irritant receptors aid in particle clearance through airway constriction and cough. The epithelium acts as a barrier to the penetration of inhaled material into the airway wall. Morphologic studies have identified the tight junctions adjoining respiratory epithelial cells as the principal barrier to passive solute translocation across the airway. New approaches have been used to study airway epithelial function. Use of excised canine trachea mounted in Ussing chambers has permitted quantitative estimates of probe molecule permeation, the correlation of permeability with bioelectric properties, and estimation of equivalent pore radii. Probe molecule diffusion across canine trachea [mean transmucosal electric potential difference (PD) = 33 mV, lumen negative] is directly related to conductance (2.9 mS/cm2) and is compatible with an equivalent pore radius of 7.5 nm. Direct measurement of tracheal PD in vivo (-29 mV) facilitates study of the genesis of the biopotential in intact animals. Measurement of the movement of HRP by radioimmunoassay has allowed correlation of the rate of probe flow across airway walls in vivo with ultrastructure. These approaches lend themselves to the study of pharmacologic and toxicologic effects on epithelial function. Antigen challenge, diethyl ether, and unfractionated cigarette smoke have been shown to increase epithelial permeability to HRP accompanied by ultrastructural evidence of tight junctional damage. Application of pharmacologic agents, e.g. amphotericin, ouabain, onto the respiratory epithelium induces similar changes in in vitro and in vivo PD. We conclude that techniques that have been used to measure permeability and transport in other epithelia may help elucidate modes of action of environmental agents on airways.