Local formation of the sphingomyelin metabolite sphingosine-1-phosphate (S1P) within the vascular wall has been shown to modulate vascular reactivity. In this study we investigated whether sphingosine kinase, the enzyme responsible for S1P synthesis, plays a role in muscarinic receptor-mediated NO production and vascular relaxation in different blood vessel types. For this purpose, sphingosine kinase translocation and sphingolipid-dependent NO-production after muscarinic receptor stimulation were assessed in an endothelial cell line. Furthermore, we used the sphingosine kinase inhibitor N,N-dimethylsphingosine (DMS) to investigate the role of sphingosine kinase in the relaxant responses to the muscarinic agonist methacholine (MCh) in isolated rat aorta and mesenteric arteries. Activation of M₃-receptors in an endothelial cell line induced a fast translocation of YFP-tagged sphingosine kinase-1 from the cytosol to the plasma membrane. Concomitant NO-production in this cell line was partially inhibited by DMS. Accordingly, in rat aorta the relaxant responses to MCh were attenuated in the presence of DMS, while the responses to the NO-donor sodium nitroprusside were unaltered. In contrast, DMS enhanced the relaxant responses to MCh in mesenteric artery preparations. This effect could also be observed in the presence of NO synthase and cyclooxygenase inhibitors, indicating that sphingosine kinase inhibition specifically enhanced endothelium-derived hyperpolarizing factor-mediated (i.e. non-NO and non-prostacyclin-dependent) relaxation. We conclude that sphingosine kinase differentially regulates vascular tone in different vessel types, enhancing NO-dependent vasorelaxation but counteracting EDHF-dependent vasorelaxation. This observation enhances our understanding of the complex mechanisms by which sphingolipids regulate vascular homeostasis. Moreover, a disturbed regulation of sphingolipid metabolism in the vascular wall may therefore play a role in the aetiology/pathology of disease states characterized by endothelial dysfunction.