Copyright © by The Rockefeller University PressSpatially restricted activation of signaling molecules governs critical aspects of cell migration; the mechanism by which this is achieved nonetheless remains unknown. Using time-lapse confocal microscopy, we analyzed dynamic redistribution of lipid rafts in chemoattractant-stimulated leukocytes expressing glycosyl phosphatidylinositol– anchored green fluorescent protein (GFP-GPI). Chemoattractants induced persistent GFP-GPI redistribution to the leading edge raft (L raft) and uropod rafts of Jurkat, HL60, and dimethyl sulfoxide–differentiated HL60 cells in a pertussis toxin–sensitive, actin-dependent manner. A trans-S membrane, nonraft GFP protein was distributed homogeneously in moving cells. A GFP-CCR5 chimera, which partitions in L rafts, accumulated at the leading edge, and CCR5 redistribution coincided with recruitment and activation of phosphatidylinositol-3 kinase in L rafts in polarized, moving cells. Membrane cholesterol depletion impeded raft redistribution and asymmetric recruitment of PI3K to the cell side facing the chemoattractant source. This is the first direct evidence that lipid rafts order spatial signaling in moving mammalian cells, by concentrating the gradient sensing machinery at the leading edge.Peer reviewed