The female hormone estrogen is an important factor in the regulation of airway function and inflammation, and sex differences in the prevalence of asthma are well described. Using an animal model, we determined how sex differences may underlie the development of altered airway function in response to allergen exposure. We compared sex differences in the development of airway hyperresponsiveness (AHR) after allergen exposure exclusively via the airways. Ovalbumin (OVA) was administered by nebulization on 10 consecutive days in BALB/c mice. After methacholine challenge, significant AHR developed in male mice but not in female mice. Ovariectomized female mice showed significant AHR after 10-day OVA inhalation. ICI182,780, an estrogen antagonist, similarly enhanced airway responsiveness even when administered 1 hour before assay. In contrast, 17beta-estradiol dose-dependently suppressed AHR in male mice. In all cases, airway responsiveness was inhibited by the administration of a neurokinin 1 receptor antagonist. These results demonstrate that sex differences in 10-day OVA-induced AHR are due to endogenous estrogen, which negatively regulates airway responsiveness in female mice. Cumulatively, the results suggest that endogenous estrogen may regulate the neurokinin 1-dependent prejunctional activation of airway smooth muscle in allergen-exposed mice.