Sphingosine-1-phosphate (S1P) is a potent bioactive lipid that has been implicated in cardiovascular disease. The objective of the present study was to determine the vasoactive effects and underlying mechanisms of S1P on adult human maternal arteries. The isometric tensions of the omental and myometrial arteries isolated from normal pregnant women at term were assessed in response to incremental doses of S1P in the presence or absence of the nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). The putative involvement of Rho-associated kinases (ROCKs) in intact arteries and in those permeabilized with α-toxin, to study agonist-dependent calcium-sensitization, was assessed with the inhibitor Y27632. Real-time RT-PCR established the presence of mRNA encoding the S1P receptors (S1P₁ to ₃), previously known as endothelial differentiation gene receptors (EDG1, 3 and 5), in both artery types. S1P induced a dose-dependent increase in the isometric tension of all the arteries. Y27632 reduced constriction due to S1P in intact arteries and reduced S1P-induced sensitization of contraction to submaximal activating Ca²⁺ in permeabilized arteries. L-NAME also modulated S1P vasoactive responses in a tissue-specific manner. Two subgroups of omental arteries were identified, one of which utilizes the NO pathway. In myometrial arteries, S1P evoked oscillatory constrictions, whereas pretreatment with L-NAME resulted in only tonic constrictions of unaltered peak magnitude. The prominent vasoactive actions of S1P in the maternal arteries of pregnant women are modulated by inhibitors of ROCKs and NO bioavailability. The subtle tissue-specific functional differences in the modulation of S1P actions by NO have important implications for vascular tone regulation by this bioactive circulatory metabolite during pregnancy.