Copyright © 2014 by the American Society of Nephrology. Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, has been implicated in regulating vascular tone and participating in chronic and acute kidney injury.However, little is known about the role of S1P in the renalmicrocirculation. Here, we directly assessed the vasoresponsiveness of preglomerular and postglomerular microvascular segments to exogenous S1P using the in vitro blood-perfused juxtamedullary nephron preparation. Superfusion of S1P (0.001-10 mM) evoked concentration-dependent vasoconstriction in preglomerular microvessels, predominantly afferent arterioles. After administration of 10 mM S1P< the diameter of afferent arterioles decreased to 35%65% of the control diameter, whereas the diameters of interlobular and arcuate arteries declined to 50%612% and 68%66% of the control diameter, respectively. Notably, efferent arterioles did not respond to S1P. The S1P receptor agonists FTY720 and FTY720-phosphate and the specific S1P1 receptor agonist SEW2871 each evoked modest afferent arteriolar vasoconstriction. Conversely, S1P2 receptor inhibition with JTE-013 significantly attenuated S1P-mediated afferent arteriolar vasoconstriction. Moreover, blockade of L-type voltage-dependent calcium channels with diltiazem or nifedipine attenuated S1P-mediated vasoconstriction. Intravenous injection of S1P in anesthetized rats reduced renal blood flow dose dependently. Western blotting and immunofluorescence revealed S1P1 and S1P2 receptor expression in isolated preglomerularmicrovessels and microvascular smooth muscle cells. These data demonstrate that S1P evokes segmentally distinct preglomerular vasoconstriction via activation of S1P1 and/or S1P2 receptors, partially via L-type voltagedependent calcium channels. Accordingly, S1P may have a novel function in regulating afferent arteriolar resistance under physiologic conditions.