Sphingosine-1 phosphate (S1P), a bioactive sphingolipid involved in diverse biological processes, is generated by sphingosine kinase (SphK) and acts via intracellular and/or extracellular mechanisms. We used biochemical, pharmacological and physiological approaches to investigate in rat myometrium the contractile effect of exogenous S1P and the possible contribution of SphK in endothelin-1 (ET-1)-mediated contraction. S1P stimulated uterine contractility (EC₅₀ = 1 µM and maximal response = 5 µM) by a pertussis toxin (PTX) insensitive and a phospholipse C (PLC) independent pathway. Phosphorylated FTY720, which interacts with all S1P receptors, excepted S1P2, failed to mimic S1P contractile response indicating that the effects of S1P involved S1P2 receptors which are expressed in myometrium. Contraction mediated by S1P and ET-1 required extracellular calcium and Rho kinase activation. Inhibition of SphK reduced ET-1-mediated contraction. ET-1, via ETA receptors coupled to PTX insensitive G proteins, stimulated SphK1 activity and induced its translocation to the membranes. Myometrial contraction triggered by ET-1 is consecutive to the sequential activation of PLC, PKC, SphK1 and Rho kinase. Prolonged exposure of the myometrium to S1P down regulated S1P2 receptors and abolished the contraction induced by exogenous S1P. However, in these conditions, the tension triggered by ET-1 was not reduced indicating that SphK activated by ET-1 contributed to its contractile effect via a S1P2 receptor independent process. Our findings demonstrated that exogenous S1P and SphK activity regulated myometrial contraction and may be of physiological relevance in the regulation of uterine motility during gestation and parturition