A successful execution and balance of adaptive immune responses requires a controlled positioning and navigation of dendritic cells (DC) into and inside secondary lymphoid organs. Whereas mechanisms were identified governing the migration of DC from peripheral nonlymphoid organs into their draining lymph nodes, little is known about the molecular cues controlling the proper positioning of spleen or lymph node resident DC. In this study, we show that the sphingosine-1 phosphate (S1P) receptor 1 influences the positioning of immature DC inside the murine spleen. Following treatment with FTY720 or SEW2871, drugs known to interfere with S1P(1)-mediated signaling, the 33D1(+) DC subpopulation homogeneously redistributes from the bridging channels to the marginal zone. In contrast, the CD205(+) DC subset remains associated with the T cell zone. Upon in vivo LPS treatment, the maturing DC assemble in the T cell zone. The LPS-driven redistribution occurs in the absence of CCR7 and cannot be prevented by FTY720, indicating that guiding mechanisms differ between immature and mature DC. Along with the observed DC subtype-specific S1P receptor expression pattern as well as the profound up-regulation of S1P(1) and S1P(3) accompanying DC maturation, these results suggest a decisive contribution of S1P signaling to intrasplenic DC motility and migration.