Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that plays important roles in allergic responses, including asthma. S1P acts on many cell types, such as mast cells, the airway epithelium, airway smooth muscle, and many immune cells. In this study we have evaluated whether a systemic administration of S1P to Balb/c mice modifies airway reactivity. Our data show that S1P (0.1-10 ng) given subcutaneously to Balb/c mice causes a specific and dose-dependent increase in cholinergic reactivity of bronchial tissues in vitro. This effect is (1) dose dependent, with a maximal effect of the dose of 10 ng of S1P; and (2) time dependent, reaching a maximal effect 21 days after S1P administration. Similarly, in the whole lung assay there is a dose- and time-dependent increase in lung resistance. Lungs isolated from S1P-treated mice displayed an increase in mast cell number. Furthermore, there is an increase of IL-4, IL-13, and IL-17 production. In conclusion, our data demonstrate that S1P signaling is involved in the complex pathway underlying airway hyperresponsiveness.