Acute exacerbations of asthma represent a common clinical problem with major economic impact. Air pollutants including ozone have been shown to contribute to asthma exacerbation, but the mechanisms underlying ozone-induced asthma exacerbation are only partially understood. The present study aimed to develop a mouse model to gain insight into the development of airway hyperresponsiveness (AHR) to methacholine (MCh) in mice after exposure to both allergen and ozone. Mice were exposed for 20 min per day for 10 consecutive days to an aerosol of 1% ovalbumin (OVA) or saline followed by a single 3-h exposure to clean air or 100, 250, or 500 ppb ozone. Ozone induced AHR in mice previously exposed to OVA when compared to non-exposed (saline) control mice. After a 10-d exposure to OVA, a single exposure to a low (100 ppb) ozone concentration was sufficient to induce AHR. The AHR response was associated with goblet-cell metaplasia. Even the lowest concentration of ozone tested, 100 ppb, which may be exceeded in urban environments and in the workplace, resulted in a significant increase in AHR, most prominent 24 h after exposure in the OVA-exposed mice.