The severity of asthma, a disease characterized by airway hyperresponsiveness and inflammation, is enhanced in some women during the menstrual cycle and during pregnancy but relieved in others. These clinical findings suggest that sex steroids modulate airway tone. Based on well-known relaxant effects of estrogens on vascular smooth muscle, we hypothesized that estrogens relax airway smooth muscle (ASM), thus facilitating bronchodilation. In ASM tissues from female patients, Western and immunocytochemical analyses confirmed the presence of both estrogen receptor (ER) isoforms, ERα and ERβ. In fura 2-loaded, dissociated ASM cells maintained in culture, acute exposure to physiological concentrations of 17β-estradiol (E₂; 100 pM to 10 nM) decreased the intracellular Ca²⁺ ([Ca²⁺]_i) response to 1 μM histamine, an effect reversed by the ER antagonist ICI-182,780. The ERα-selective agonist (R,R)-THC had a greater reducing effect on [Ca²⁺]_i responses to histamine and 1 μM ACh compared with the ERβ-selective agonist (DPN). The effects of E₂ on [Ca²⁺]_i were mediated, at least in part, via decreased Ca²⁺ influx through l-type channels and store-operated Ca²⁺ entry but not via Ca²⁺-activated K⁺ channels, receptor-operated entry, or sarcoplasmic reticulum reuptake. Overall, these data support our hypothesis that estrogens relax ASM and suggest a potentially novel therapeutic target in airway hyperresponsiveness.