We characterized the effect of Sphingosine-1-phosphate (S1P) on vascular tone. S1P selectively constricted isolated cerebral, but not peripheral arteries, despite ubiquitous expression of S1P₁, S1P₂, S1P₃ and S1P₅ receptor mRNA. Clostridium B and C3 toxins and the rho-kinase inhibitor Y27632 (trans-N-(4-pyridyl)-4-(l-aminoethyl)-cyclohexane carboxamide) reduced this vasoconstriction to S1P, indicating that the response was mediated through Rho. Pertussis toxin displayed only weak inhibition, suggesting minor involvement of G_i/o protein. The S1P effect was specifically reduced by adenovirus bearing a s1p₃ but not s1p₂, antisense construct. Furthermore, suramin, which selectively blocks S1P₃ receptors, inhibited the vasoconstrictor effect of S1P, indicating that S1P₃ receptors account for at least part of S1P-mediated vasoconstriction in cerebral arteries. In vivo, intracarotid injection of S1P decreased cerebral blood flow, an effect prevented by suramin treatment. Because S1P constricts cerebral blood vessels and is released from platelets during clotting, the S1P/S1P₃ system constitutes a novel potential target for cerebrovascular disease therapy.