The female hormone estrogen is an important factor in the regulation of airway function and inflammation and gender differences in the prevalence of asthma are well-described. Using an animal model, we determined how gender differences may underlie the development of altered airway function in response to allergen exposure. We compared gender 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. Following methacholine challenge, significant AHR only developed in male mice but not in female mice. However, ovariectomized female mice showed significant AHR after 10-day OVA inhalation; an estrogen antagonist, similarly enhanced airway responsiveness, even when administered 1 hr prior to assay. In contrast, 17-estradiol dose-dependently suppressed AHR in male mice. In all cases, airway responsiveness was inhibited by administration of a neurokinin 1 (NK-1) receptor antagonist. These results demonstrate that gender differences in 10-day OVA-induced AHR are due to endogenous estrogen which negatively regulates airway responsiveness in female mice. Cumulatively, the results suggest endogenous estrogen may regulate the NK-1-dependent pre-junctional activation of airway smooth muscle in allergen-exposed mice.