Gammadelta T cells may contribute to the pathogenesis of Multiple Sclerosis (MS) via cytotoxicity directed at the myelin-oligodendrocyte unit. We have previously demonstrated that peripheral blood-derived gammadelta T cells lyse fresh human oligodendrocytes in vitro. The present work extends these observations to gammadelta T cells derived from both peripheral blood (PBL) and cerebrospinal fluid (CSF) of MS and non-MS neurological disease controls and addresses the mechanism of cellular cytotoxicity. We found that MS patients contained increased proportions of Vdelta1+ gammadelta T cells in both CSF and PBL samples compared to other neurological disease (OND) controls. Although gammadelta T cells from all patients were cytotoxic towards Daudi, RPMI 8226, U937, Jurkat, oligodendroglioma and fresh human oligodendrocyte targets, OND-derived, Vdelta2+ rich, populations derived from the CSF exhibited greater cytotoxicity towards cell lines (Daudi, RPMI 8226) known to express high levels of heat shock proteins (hsp). To clarify the mechanism(s) of cytotoxicity used by gammadelta T cells, we first showed that cell-target contact was necessary by the use of physical barriers (transwells), which reduced target cell lysis by at least 75%. The use of Ca2+-free media reduced lysis by up to 50%, but fully blocking gammadelta T cell Perforin release and function by either Ca2+ chelation (Mg2EGTA) or the H+-ATPase inhibitor Concanamycin-A (CMA), completely abrogated the lysis of Fas-/hsp60high expressing targets (Daudi, U937). However, additional treatment with Brefeldin A was required for the complete inhibition of gammadelta T cell mediated killing of Fas+ expressing Jurkat targets and fresh human brain-derived oligodendrocytes. Inhibition of granzyme activity by an isocoumarin compound reduced cytolysis only slightly. The use of either Brefeldin A or an anti-Fas antibody alone did not significantly affect lysis. These findings suggest that in MS, gammadelta T cells may utilize either the Fas-mediated or Perforin-based cell cytotoxicity pathways in exerting oligodendrocyte damage, though the Perforin pathway is predominant.