Interleukin (IL)-10 is an anti-inflammatory cytokine implicated in the regulation of airway inflammation in asthma. Among other activities, IL-10 suppresses production of NO; consequently, its absence may permit increased NO production that can affect airway smooth muscle contractility. Therefore, we investigated airway reactivity (AR) in response to methacholine (MCh) in IL-10 knockout (^-/-) mice as compared to wild-type C57Bl6 (C57) mice, in which airway NO production was measured as exhaled NO (E_NO), and NO production was altered with administration of either NO synthase (NOS)-specific inhibitors, or recombinant murine (rm)IL-10. AR, measured as Penh in vivo, and tracheal ring tension in vitro, was lower in IL-10^-/- mice by 25-50%, which was associated with elevated E_NO levels (13 vs. 7 ppb). Administration of NOS inhibitors N^G-nitro-L-arginine-methyl ester (L-NAME; 8 mg/kg, i.p.) or L-N⁶-(1-iminoethyl)-lysine (L-NIL; 3 mg/kg, i.p.) to IL-10-/- mice decreased ENO by an average of 50%, which was associated with increased AR, to levels similar to C57 mice. E_NO in IL-10^-/- mice decreased in a dose-dependent fashion in response to administered rmIL-10, to levels similar to C57 mice (7 ppb), which was associated with a 30% increment in AR. Thus, increased NO production in the absence of IL-10, decreased AR, which was reversed with inhibition of NO, either by inhibition of NOS, or with reconstitution of IL-10. These findings suggest that airway NO production can modulate airway smooth muscle contractility, resulting in airway hyporesponsiveness when IL-10 is absent.