Sphingosine 1-phosphate (S1P) is a biologically active lipid. In vitro, S1P tightens the endothelial barrier, as assessed by a rapid increase in electrical resistance and a decrease in solute permeability. We hypothesized that this activity of S1P would also occur in vivo. Hydraulic conductivity (L_p), an assessment of endothelial barrier function, was measured in individually perfused venules in rat mesenteries. S1P (1 µM) decreased basal L_p by 63% when basal L_p was between 3.6 and 4.1 x 10^-7 cm^.sec^-1[[rad]]cm H₂O^-1 but showed no effect when basal L_p was below 2 x 10^-7 cm^.sec^-1.cm H₂O^-1. Under either condition, S1P blocked the 6-fold increase in L_p induced by platelet activating factor (PAF, 10 nM). Perfusion of venules with pertussis toxin (0.1 µg/ml) for 3 h did not affect basal L_p or the increased L_p induced by PAF but significantly attenuated the inhibitory action of S1P on the PAF-induced increase in L_p, indicating the involvement of the inhibitory G protein, G_i. Measurement of endothelial [Ca²⁺]_i in venules loaded with Fura-2-AM showed that S1P alone transiently increased basal endothelial [Ca²⁺]_i (from 89 nM to 193 nM) but had no effect on the magnitude and time course of the PAF-induced increase in endothelial [Ca²⁺]_i. These results indicate that S1P functions in vivo to prevent the PAF-induced increase in microvessel permeability. The inhibitory action of S1P involves the pertussis toxin-sensitive G_i protein and is not mediated by prevention of the PAF-induced increase in endothelial [Ca²⁺]_i.