Sphingosine-1-phosphate (S1P) has been demonstrated to enhance endothelial barrier function in vivo and in vitro. However, different S1P receptor subtypes have been indicated to play different or even opposing roles in the regulation of vascular barrier function. This study aims to differentiate the roles of endogenous endothelial S1P subtype receptors in the regulation of permeability in intact microvessels using specific receptor agonist and antagonists. Microvessel permeability was measured with hydraulic conductivity ( L_p) in individually perfused rat mesenteric venules. S1P-mediated changes in endothelial intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured in fura-2-loaded venules. Confocal images of fluorescent immunostaining illustrated the spatial expressions of three S1P subtype receptors (S1P_R1–3) in rat venules. The application of S1P (1 μM) in the presence of S1P_R1–3 inhibited platelet-activating factor- or bradykinin-induced permeability increase. This S1P effect was reversed only with a selective S1P_R1 antagonist, W-146, and was not affected by S1P_R2 or S1P_R3 antagonists JTE-013 and CAY-10444, respectively. S1P_R1 was also identified as the sole receptor responsible for S1P-mediated increases in endothelial [Ca²⁺]_i. S1P_R2 or S1P_R3 antagonist alone affected neither basal L_p nor platelet-activating factor-induced permeability increase. The selective S1P_R1 agonist, SEW-2871, showed similar [Ca²⁺]_i and permeability effect to that of S1P. These results indicate that, despite the presence of S1P_R1–3 in the intact venules, only the activation of endothelial S1P_R1 is responsible for the protective action of S1P on microvessel permeability and that endogenous S1P_R2 or S1P_R3 did not exhibit functional roles in the regulation of permeability under basal or acutely stimulated conditions.