We studied the role of the guinea pig tracheal epithelium in modulating tracheal smooth muscle responses to the relaxant agonists albuterol, sodium nitroprusside, and theophylline. We used an in vitro preparation that allowed separation of the fluids bathing the luminal (internal) and serosal (external) surfaces of the trachea, and bronchodilators were administered to either surface of carbachol-contracted tracheae. All three drugs produced dose-dependent relaxation. However, albuterol and nitroprusside were less potent (concentration that produced half-maximal effect increased by 100- and 32-fold, respectively) when given to the epithelial side with the epithelium intact compared with the epithelium denuded or compared with serosal administration with the epithelium intact. These differences were not observed for theophylline, where smooth muscle responses were independent of either the side of stimulation or of the presence or absence of the epithelium. Direct measurements of the diffusion of theophylline across the tracheal wall in the presence or absence of epithelium showed that after 5 h of incubation with a fixed luminal concentration of theophylline, only 1.7% had diffused across the tracheal wall with the epithelium intact. This increased to only approximately 3.3% when the epithelium was denuded. These results suggest that the epithelial is a relatively weak barrier for lipophilic agents but has a major role as a diffusion barrier to hydrophilic substances.