While airway constriction has been shown to affect exhaled nitric oxide (NO), the mechanisms and location of constricted airways most likely to affect exhaled NO remain obscure. We studied the effects of histamine-induced airway constriction and ventilation heterogeneity on exhaled NO at 50ml/s (FENO₅₀), and combined this with model simulations of FENO₅₀ changes due to constriction of airways at various depths of the lung model. In twenty normal subjects, histamine induced a 26+15(SD)% FENO₅₀ decrease, a 9+6% FEV₁ decrease, a 19+9% FEF_25-75 decrease and a 94+119% increase in conductive ventilation heterogeneity. There was a significant correlation of FENO₅₀ decrease with FEF_25-75 decrease (p=0.006), but not with FEV₁ decrease nor with increased ventilation heterogeneity. Simulations confirmed the negligible effect of ventilation heterogeneity on FENO₅₀ and showed that the histamine-induced FENO₅₀ decrease was due to constriction, with associated reduction in NO flux, of airways located proximal to generation 15. The model also indicated that the most marked effect of airways constriction on FENO₅₀ is situated in generation 10-15 and that airway constriction beyond generation 15 markedly increases FENO₅₀, due to interference with the NO back diffusion effect. These mechanical factors should be considered when interpreting exhaled NO in lung disease.