Gammadelta T lymphocytes are thought to be involved in multiple sclerosis (MS) pathogenesis. In this work, we discuss the characteristics of these cells and possible implications in the pathogenesis of MS, focusing on the mechanism(s) underlying extravasation and tissue localization. Phenotype and transendothelial migration of γδ T cells from healthy donors and patients with relapsing-remitting MS were studied. In MS patients the Vδ2 T cell subset, expressing NKRP1A/CD161 adhesion molecule, is expanded and capable of transendothelial migration. Vδ1/Vδ2 subsets use distinct signal transduction pathways: Vδ1 cells lack NKRP1A and express PECAM-1/CD31, which drives transmigration, while Vδ2 cells are PECAM-1 negative and use NKRP1A. Vδ2 migration is coupled with CAMKII, whereas Vδ1 depend on PI-3K. NKRP1A and PECAM-1 selectively activate the two pathways: indeed, oligomerization of NKRP1A on Vδ2 T cells leads to CAMKII activation, occupancy of PECAM-1 on Vδ1 cells triggers the PI-3K-dependent Akt/PKB pathway. Moreover, Vδ2 T cells are CXCR3 brightCXCR4dull, while Vδ1 are mostly CXCR4 +. Vδ1 and Vδ2 cells transmigrate in response to IP-10/CXCL10 and SDF-1/CXCL12 according to the expression of their specific receptors. In a fraction of Vδ1 T cells coexpressing CXCR3 and CXCR4, the homeostatic chemokine 6Ckine/SLC/CCL21 is more effective. IP-10/CXCL10 or 6Ckine/SLC/CCL21 and SDF-1/CXCL12-induced transmigration is coupled to PI-3K/Akt/PKB, but only CXCR3 is capable of inducing CAMKII activation. We suggest that both subsets of γδ T lymphocytes may migrate to the site of lesion in MS using two different signaling pathways to extravasate and responding to different chemokines.