Aims: Sphingosine-1-phosphate (S1P) has emerged as a potent bioactive lipid with multiple functions in cardiovascular pathophysiology. Potential roles of S1P in heart valve diseases and expression of relevant receptors (S1P1, S1P2, or S1P3) in valve tissue and in valvular interstitial cells (VICs), the major cell population with essential functions in maintenance of valvular structure, are currently unknown. Methods and results: Exposure to S1P (622000 nM) of cultured VICs from porcine aortic valves on cell culture polystyrene resulted in contraction and nodule formation. The S1P-dependent contraction was completely inhibited by blockers of S1P2, RhoA, and RhoA-associated protein kinase (ROCK). Activated RhoA was clearly increased after S1P treatment, whereas activated Rac1 was only slightly reduced. In addition, exposure to S1P induced a transient increase in cytosolic Ca ²⁺. Application of channel blockers and other effectors of Ca ²⁺ homeostasis showed that the S1P effect is largely caused by Ca ²⁺ release from internal stores. However, resistance to blocking S1P2, different kinetics, as well as concentration dependence exclude a major role of Ca ²⁺ influx in S1P-induced nodule formation. In order to verify the effects in situ, contractions of valve tissue slices were measured. The S1P-induced isometric contraction of valve leaflets was of similar force amplitude as observed with adrenaline. The effect was fully reversed by blocking S1P2. Conclusion: The results suggest that S1P induces contraction of VICs from porcine aortic valves by signalling via S1P2, RhoA, and ROCK. In this way, S1P may contribute to regulation of tissue tension in aortic valves.