Cyclic ADP-ribose (cADPR) mobilizes calcium from intracellular stores and contributes to agonist-induced intracellular calcium elevation in airway smooth muscle (ASM). In this study we determined the functional role of CD38/cADPR signaling in the regulation of airway tone using CD38 deficient (cd38 -/-) mice. The responsiveness to different doses of methacholine, as determined by changes in lung resistance and dynamic compliance, was significantly (P ≤ 0.05) lower in cd38-/- mice compared with wild-type controls. To determine the mechanism responsible for the reduced responsiveness, we measured the intracellular calcium responses to contractile agonists in ASM cells. In ASM cells isolated from cd38-/- mice, the intracellular calcium responses to acetylcholine and endothelin-1 were significantly lower than in controls. Pretreatment of ASM cells with a cADPR antagonist resulted in attenuated intracellular calcium responses to endothelin-1 in cells isolated from wild-type mice, but not in those isolated from the cd38-/- mice. Very low cADPR levels and no detectable ADP-ribosyl cyclase activity were observed in lung tissue from cd38 -/- mice, suggesting that CD38 is a critical source for cADPR synthesis. The results of the present study demonstrate that CD38/cADPR contributes to airway smooth muscle tone and responsiveness through its effects on agonist-induced elevation of intracellular calcium in ASM cells.