The purpose of the study was to evaluate the importance of the epithelium in determining the potency of exogenous vasoactive intestinal peptide (VIP) in inhibiting responses of isolated guinea pig trachea to vagal stimulation. Isolated innervated tracheal preparations (n = 56) were mounted in glass organ baths in Krebs-Henseleit (K-H) solution at 37 degrees C and gassed with 95% O2-5% CO2. The inside of the trachea was separately perfused with K-H solution at 1 ml/min. The vagal nerve trunks were stimulated (20 V, 1-ms pulses, 10-s trains) at low (0.5 Hz) and high frequency (15 Hz) alternately, and the contractile responses were measured as increases in intratracheal pressures. VIP (10(-8)-10(-7) M) inhibited responses to both high- and low-frequency stimulation. VIP was more potent in inhibiting contractions when administered to the outside than the inside surface of the trachea, and disruptionon of the epithelium abolished this difference. The endopeptidase inhibitors phosphoramidon and thiorphan (5 x 10(-6) M) potentiated the action of VIP. These data indicate that the epithelium reduces the efficacy of VIP. We suggest that the epithelium is a site of degradation of VIP by endopeptidase and may also be a diffusion barrier.