Microglia motility plays a crucial role in response to lesion or exocytotoxic damage of the cerebral tissue. The neuropeptide neurotensin elicited the migration of the human microglial cell line C13NJ by a mechanism dependent on both phosphatidylinositol-3 kinase (PI3 kinase) and mitogen-activated protein (MAP) kinases pathways. The effect of neurotensin on cell migration was blocked by the neurotensin receptor-3 propeptide, a selective ligand of this receptor. The type I neurotensin receptor-3 was the only known neurotensin receptor expressed in these microglial cells, and its activation led to the phosphorylation of both extracellular signaling-regulated kinases Erk1/2 and Akt. Furthermore, the effect of neurotensin on cell migration was preceded by a profound modification of the F-actin cytoskeleton, particularly by the rapid formation of numerous cell filopodia. Both the motility and the filopodia appearance induced by neurotensin were totally blocked by selective inhibitors of MAP kinases or PI3 kinase pathways. In the murine microglial cell line N11, the neurotensin receptor-3 is also the only neurotensin receptor expressed, and its activation by neurotensin leads to the phosphorylation of both Erk1/2 and Akt. In these cells, neurotensin induces the gene expression of several cytokines/chemokines, including MIP-2, MCP-1, interleukin-1beta and tumor necrosis factor-alpha. This induction is dependent on both protein kinases pathways. We observed that the effect of neurotensin on the cytokine/chemokine expression is also inhibited by the neurotensin receptor-3 propeptide. This is the demonstration that the neurotensin receptor-3 is functional and mediates both the migratory action of neurotensin and its induction of chemokines/cytokines expression.