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Lysosulfatide is a derivative of the glycosphingolipid sulfatide. It is a major component of high density lipoproteins and was detected in the human brain. Here, we show that lysosulfatide acts as an extracellular signal regulating the migration of a neural precursor cell line (B35 neuroblastoma cells) by rapidly promoting process retraction and cell rounding. These cells express the lysosulfatide receptor S1P3 according to RT-PCR, western blotting and immunocytochemistry, but S1P3 does not mediate the effect since preincubation with three different compounds known to inhibit S1P3 did not block lysosulfatide-induced cell rounding. The signal transduction after stimulation with 3 microM lysosulfatide involves a rapid increase of [Ca2+]i which causes process retraction. This mechanism may be relevant under conditions where neural cells encounter elevated lysosulfatide levels as for example under pathological conditions after breakdown of the blood brain barrier or possibly in the lysosomal sulfatide storage disorder metachromatic leukodystrophy.