While gender differences incidence and severity of asthma are clinically recognized, the physiological role of sex steroids is less clear, with cyclical exacerbations (pre-menstrual asthma) occurring in late luteal phase when estrogen levels are low, while high sex steroid levels (e.g. pregnancy) are not always associated with worsening of symptoms. Similar to endothelium in vasculature, airway epithelium is a source of bronchodilatory agents such as NO. Using human bronchial rings and epithelial cells derived from such samples (obtained from surgery), we tested the hypothesis that estrogens facilitate bronchodilation by enhancing NO production in airway epithelium, resulting in reduced intracellular Ca²⁺ ([Ca²⁺]_i) in airway smooth muscle. Values are means with SE. Acute exposure to 1 nM 17β-estradiol (E₂) relaxed epithelium-intact bronchial rings contracted with 1 μM ACh to a significantly greater extent (72 ± 9% of max. force) than epithelium-denuded rings (42 ± 8%; p<0.05, n=4, t-test). E₂-induced relaxation was blunted by the NO scavenger PTIO (48 ± 9%). In isolated human airway epithelial cells (n=20 per group) loaded with the fluorescent NO indicator DAF-2, 1 nM E₂ increased fluorescence levels (28 ± 3 arbitrary units (au) with 5 ± 2 au baseline), comparable to that induced by 1 μM ACh (32 ± 7 au) or 50 μM ATP (41 ± 4 au). In the presence of E₂, ACh effects on DAF-2 fluorescence were enhanced (69 ± 5 au), while PTIO substantially blunted these effects (22 ± 6 au). The estrogen receptor (ER) specific agonists THC (ERα) and DPN (ERα) both induced comparable NO production (31 ± 6 au vs. 38 ± 6 au). Neither ACh nor E₂ affected fluorescence of the NO-insensitive dye DAF-4. Western blot analyses of eNOS phosphorylation at Ser1177 showed an ~5-fold increase with E₂ exposure. Finally, in Transwell plates where human epithelial cells and airway smooth muscle cells were co-cultured, exposure of epithelial cells to 1 nM E₂ induced 58 ± 7% reductions in ACh-induced elevation of [Ca²⁺]_i in airway smooth muscle cells. Overall, these studies indicate a prominent role for airway epithelium and epithelium-derived NO in the bronchodilatory effects of estrogens in humans.