Airway hyperresponsiveness (AHR) is a hallmark of bronchial asthma. Important features of this exaggerated response to bronchoconstrictive stimuli have mostly been investigated in vivo in intact animals or in vitro in isolated tracheal or bronchial tissues. Both approaches have important advantages but also certain limitations. Therefore, the aim of our study was to develop an ex vivo model of isolated lungs from sensitized mice for the investigation of airway responsiveness (AR). BALB/c mice were sensitized by intraperitoneal ovalbumin (Ova) and subsequently challenged by Ova inhalation. In vivo AR was measured in unrestrained animals by whole body plethysmography after stimulation with aerosolized methacholine (MCh) with determination of enhanced pause (P(enh)). Twenty-four hours after each P(enh) measurement, airway resistance was continuously registered in isolated, perfused, and ventilated lungs on stimulation with inhaled or intravascular MCh or nebulized Ova. In a subset of experiments, in vivo AR was additionally measured in orotracheally intubated, spontaneously breathing mice 24 h after P(enh) measurement, and lungs were isolated further 24 h later. Isolated lungs of allergen-sensitized and -challenged mice showed increased AR after MCh inhalation or infusion as well as after specific provocation with aerosolized allergen. AR was increased on days 2 and 5 after Ova challenge and had returned to baseline on day 9. AHR in isolated lungs after aerosolized or intravascular MCh strongly correlated with in vivo AR. Pretreatment of isolated lungs with the beta(2)-agonist fenoterol diminished AR. In conclusion, this model provides new opportunities to investigate mechanisms of AHR as well as pharmacological interventions on an intact organ level.