Exogenous sphingosine-1-phosphate (S1P), a lipid mediator in blood, attenuates acute microvascular permeability increases via receptor S1P1 to stabilize the endothelium. To evaluate the contribution of erythrocytes as an endogenous source of S1P to the regulation of basal permeability, we measured permeability coefficients in intact individually perfused venular microvessels of rat mesentery. This strategy also enabled the contributions of other endogenous S1P sources to be evaluated. Apparent permeability coefficients (P(S)) to albumin and α-lactalbumin and the hydraulic conductivity of mesenteric microvessels were measured in the presence or absence of rat erythrocytes or rat erythrocyte-conditioned perfusate. Rat erythrocytes added to the perfusate were the principal source of S1P in these microvessels. Basal P(S) to albumin was stable and typical of blood-perfused microvessels (mean 0.5 × 10(-6) cm/s) when erythrocytes or erythrocyte-conditioned perfusates were present. When they were absent, P(S) to albumin or α-lactalbumin increased up to 40-fold (over 10 min). When exogenous S1P was added to perfusates, permeability returned to levels comparable with those seen in the presence of erythrocytes. Addition of SEW 2871, an agonist specific for S1P1, in the absence of red blood cells reduced P(S)(BSA) (40-fold reduction) toward basal. The specific S1P1 receptor antagonist (W-146) reversed the stabilizing action of erythrocytes and increased permeability (27-fold increase) in a manner similar to that seen in the absence of erythrocytes. Erythrocytes are a primary source of S1P that maintains normal venular microvessel permeability. Absence of erythrocytes or conditioned perfusate in in vivo and in vitro models of endothelial barriers elevates basal permeability.