Ozone (O₃)-induced airway hyperresponsiveness in laboratory animals is usually demonstrated through dose-response curves with inhaled or intravenous bronchoconstrictor agonists. However, comparability of these two routes has not been well documented. Thus guinea pig airway responsiveness to ACh and histamine was evaluated 16-18 h after O₃ (3 parts/million, 1 h) or air exposure by two plethysmographic methods (spontaneously breathing and mechanically ventilated) and by two administration routes (inhalatory or intravenous). We found that O₃ caused airway hyperresponsiveness to intravenous, but not to inhaled, agonists, independent of the plethysmographic method used. Suitability of the inhalatory route to detect airway hyperresponsiveness was corroborated with inhaled ACh after an antigen challenge or extending O₃ exposure to 3 h. Acetylcholinesterase activity was not modified after O₃ exposure in lung homogenates and blood samples. Thus inhaled agonists were less effective to reveal the airway hyperresponsiveness after an acute O₃ exposure than intravenous ones, at least for the 1-h exposure to 3 parts/million, and this difference seems not to be related to an O₃-induced inhibition of the acetylcholinesterase activity.