Nicotinic acid adenine dinucleotide phosphate (NAADP) is increasingly being demonstrated to be involved in calcium signaling in many cell types and species. Although it has been shown to play a role in smooth muscle cell contraction in several tissues, nothing is known about its possible role in tracheal smooth muscle, a muscle type that is clinically relevant to asthma. To determine whether NAADP functions as a second messenger in tracheal smooth muscle contraction, we used the criteria set out by Sutherland for a molecule to be designated a second messenger. We report that NAADP satisfies all five criteria as follows. First, the NAADP antagonist Ned-19 inhibited contractions in tracheal rings and calcium increases in isolated smooth muscle cells induced by the muscarinic agonist carbachol. Second, NAADP increased cytosolic calcium in isolated cells when microinjected and was blocked by Ned-19. Third, tracheal homogenates could synthesize NAADP by base exchange from exogenous NADP and nicotinic acid and metabolize exogenous NAADP to nicotinic acid adenine dinucleotide by a 2'-phosphatase. Fourth, carbachol induced a rapid and transient increase in endogenous NAADP levels. Fifth, tracheal homogenates contained NAADP-binding sites of high affinity. Taken together, these data demonstrate that NAADP functions as a second messenger in tracheal smooth muscle, and therefore, steps in the NAADP signaling pathway might provide possible new drug targets.