Marginal zones (MZs) are microdomains in the spleen that contain various types of immune cells, including MZ B cells, MOMA1(+) metallophilic macrophages, and mucosal addressin cell adhesion molecule 1 (MAdCAM-1)(+) endothelial cells. MAdCAM-1(+) and MOMA1(+) cells line the sinus, that separates MZs from splenic follicles. Here we show that a receptor for the lysophospholipid sphingosine-1-phosphate (S1P), S1P(3), is required for normal numbers of splenic immature and MZ B cells, and for S1P-induced chemotaxis of MZ B cells. S1P(3) is also essential for proper alignment of MOMA1(+) macrophages and MAdCAM-1(+) endothelial cells along the marginal sinus. The lack of cohesion of the marginal sinus in S1P(3)(-/-) mice affects MZ B cell functions, as wild-type (WT) MZ B cells migrate more into S1P(3)(-/-) follicles than into WT follicles after treatment with lipopolysaccharide. Additionally, short-term homing experiments demonstrate that WT MZ B cells home to the S1P(3)(-/-) spleen in increased numbers, suggesting a role for the marginal sinus in regulating MZ B cells numbers. Moreover, S1P(3)(-/-) mice are defective in mounting immune responses to thymus-independent antigen type 2 due to defects in radiation-resistant cells in the spleen. These data identify lysophospholipids and the S1P(3) receptor as essential regulators of the MZ sinus and its role as a barrier to the follicle.