We examined the hypothesis that, after ozone (O3) inhalation exposure, changes in the production of EpDRF by respiratory epithelium could affect reactivity of guinea pigs to inhaled methacholine (MCh). Dose-response curves for enhanced pause (Penh) responses to inhaled MCh were obtained from conscious animals before, immediately after, and 24 hr after inhalation of 3 ppm O3, for 1 hr. Airway reactivity in vitro was examined using the perfused trachea preparation to apply MCh separately to the serosal surface (extraluminal (EL) bath) or to the mucosal surface (intraluminal (IL) perfusing perfusate); EpDRF release was examined using IL NaCl-stimulated relaxant responses of MCh-contracted preparations. Immediately after O3 -exposure, reactivity to inhaled MCh was significantly increased, whereas by 24 hr reactivity declined to the control level. Immediately after O3, delivery, reactivity to IL MCh was increased in the presence but not in the absence of the epithelium, and responsiveness to EL MCh was unchanged; by 24 hr post-exposure reactivity to IL MCh had returned to the control level. Immediately after O3 exposure, IL NaCl-induced, EpDRF-mediated relaxation responses were inhibited, but by 24 hr responses to IL NaCl were normal. Throughout the post-exposure periods the epithelium was substantially damaged. These findings indicate that the development of airway hyperreactivity to inhaled MCh occurs in association with decreased EpDRF release, whereas recovery to normal reactivity is accompanied by the return of EpDRF release to normal.