Gender differences in incidence and severity of asthma are clinically observed. Pre-menstrual exacerbations are inconsistent and actually occur in late luteal phase when estrogen levels are lowest while in spite of high sex steroid levels, pregnancy is not always associated with worsening of symptoms. Diseases such as asthma are characterized by airway hyperresponsiveness that involves altered intracellular Ca²⁺ ([Ca²⁺]_i) responses of airway smooth muscle (ASM) to agonist stimulation. In this regard, we have recently demonstrated an important role for caveolae in [Ca²⁺]_i regulation. In vascular endothelium, estrogen receptors (ERs) are expressed within caveolae, and facilitate vasodilation. We hypothesized that estrogens facilitate bronchodilation by decreasing [Ca²⁺]_i in ASM. In ASM isolated from human lung surgical waste tissue, we found through Western blot analysis (n=4) that both estrogen receptors (ERs), ERα and ERβ are expressed to substantial extents, as are two truncated ER isoforms (ERα-36 and ERα-47). ERα is also expressed within caveolae. Overnight exposure to 20 ng/ml TNFα substantially increased caveolar ERα (and to a lesser extent ERβ) expression. In ASM cells loaded with the Ca²⁺ indicator fura-2, acute exposure to 17β-estradiol (17βE₂; 1nM) blunted [Ca²⁺]_i response to bronchoconstrictor agonists (1 μM ACh, 10 μM histamine) by ~60% of peak Ca²⁺ responses. These effects were prevented by pre-exposure to ER antagonist (ICI 182,780; 20% of peak responses). Overnight exposure to TNFα enhanced E₂ effects on [Ca²⁺]_i by ~50%. Pre-exposure to the cholesterol chelator methyl-β-cyclodextrin (10 mM) blunted E₂ effects (35% of peak responses). These novel data suggest that in human ASM, estrogens act via ERs (potentially within caveolae) to non-genomically produced bronchodilation. Given enhanced ER expression with inflammation, estrogens may be a novel therapeutic mechanism in airway diseases such as asthma.