Sphingosine-1-phosphate (S1P) acts as an extracellular ligand for a family of G-protein coupled receptors that are crucial in cell migration. S1P5 is exclusively expressed in oligodendrocytes and oligodendrocyte precursor cells (OPCs), which migrate considerable distances during brain development. The current studies suggest a physiological role for S1P and S1P5 in regulation of OPC migration. mRNA expression levels of S1P2 and S1P5 are comparable in OPCs, but S1P binding specifically to the S1P5 receptor blocked OPC migration (IC50=29 nM). Thus, knocking down S1P5 using siRNA prevented the S1P-induced decrease in OPC migration, whereas knocking down S1P2 did not have any effect. S1P-induced modulation of OPC migration was insensitive to pertussis toxin, suggesting that S1P5-initiated signaling is not mediated by the G alpha(i)-protein coupled pathway. Furthermore, S1P5 appears to engage the G alpha(12/13) protein coupled Rho/ROCK signaling pathway to impede OPC migration. To modulate OPC motility, extracellular S1P could be derived from the export of intracellular S1P generated in response to glutamate treatment of OPCs. These studies suggest that S1P could be a part of the neuron-oligodendroglial communication network regulating OPC migration and may provide directional guidance cues for migrating OPCs in the developing brain.