Distinct subsets of dendritic cells (DCs) are present in blood, probably “en route” to different tissues. We have investigated the chemokines and adhesion molecules involved in the migration of myeloid (CD11c⁺) and plasmacytoid (CD123⁺) human peripheral blood DCs across vascular endothelium. Among blood DCs, the CD11c⁺ subset vigorously migrated across endothelium in the absence of any chemotactic stimuli, whereas spontaneous migration of CD123⁺ DCs was limited. In bare cell migration assays, myeloid DCs responded with great potency to several inflammatory and homeostatic chemokines, whereas plasmacytoid DCs responded poorly to all chemokines tested. In contrast, the presence of endothelium greatly favored transmigration of plasmacytoid DCs in response to CXCL12 (stromal cell-derived factor-1) and CCL5 (regulated on activation, normal T expressed and secreted). Myeloid DCs exhibited a very potent transendothelial migration in response to CXCL12, CCL5, and CCL2 (monocyte chemoattractant protein-1). Furthermore, we explored whether blood DCs acutely switch their pattern of migration to the lymph node-derived chemokine CCL21 (secondary lymphoid-tissue chemokine) in response to microbial stimuli [viral double-stranded (ds)RNA or bacterial CpG-DNA]. A synthetic dsRNA rapidly enhanced the response of CD11c⁺ DCs to CCL21, whereas a longer stimulation with CpG-DNA was needed to trigger CD123⁺ DCs responsive to CCL21. Use of blocking monoclonal antibodies to adhesion molecules revealed that both DC subsets used platelet endothelial cell adhesion molecule-1 to move across activated endothelium. CD123⁺ DCs required β₂ and β₁ integrins to transmigrate, whereas CD11c⁺ DCs may use integrin-independent mechanisms to migrate across activated endothelium.