Sphingosine 1-phosphate type 1 (S1P₁) receptor agonists cause sequestration of lymphocytes in secondary lymphoid organs by a mechanism that is not well understood. One hypothesis proposes that agonists act as 'functional antagonists' by binding and internalizing S1P₁ receptors on lymphocytes; a second hypothesis proposes instead that S1P₁ agonists act on endothelial cells to prevent lymphocyte egress from lymph nodes. Here, two-photon imaging of living T cells in explanted lymph nodes after treatment with S1P₁ agonists or antagonists has provided insight into the mechanism by which S1P₁ agonists function. The selective S1P₁ agonist SEW2871 caused reversible slowing and 'log-jamming' of T cells between filled medullary cords and empty sinuses, whereas motility was unaltered in diffuse cortex. Removal or antagonist competition of SEW2871 permitted recovery of T cell motility in the parenchyma of the medulla and resumption of migration across the stromal endothelial barrier, leading to refilling of sinuses. Our results provide visualization of transendothelial migration of T cells into lymphatic sinuses and suggest that S1P₁ agonists act mainly on endothelial cell S1P₁ receptors to inhibit lymphocyte migration.