Background: Fractional exhaled nitric oxide measured at expiratory flow of 50 mL/s (FENO50), a biomarker of airway inflammation, is affected by changes in airway caliber. Whether a lower FENO50 level during bronchoconstriction is only an artifact due to the strong flow dependence of this parameter is controversial. Methods: We aimed to evaluate the dynamics of airway and alveolar nitric oxide (NO) during acute bronchoconstriction induced by methacholine. Exhaled NO was measured at expiratory flows of 10, 50, 100, 150, and 250 mL/s before and after metacholine in 26 responders to methacholine and 37 nonresponders. Flow-independent parameters (airway wall NO flux, airway NO diffusing capacity, airway wall NO concentration, alveolar NO concentration) were calculated using a two-compartment model, and correction for NO axial back diffusion was applied. Results: Bronchoconstriction in responders was associated with a decrease in FENO50 (-28%, P