NKT cells constitute a small (<0.1% of blood and marrow T cells) but potent subset of regulatory T cells. Upon engagement of their unique TCR by the glycolipid presenting, MHC-like, non-polymorphic molecule CD1d, they are activated and secrete copious amounts of TH1 and TH2 cytokines. Activated NKT have a pivotal role in modulating all aspects of the innate and adaptive immune responses mainly through their interaction with antigen presenting cells (APC). Mice deficient in NKT or CD1d have diminished TH1 responses against a variety of pathogens and tumours. Conversely, administration of the model glycolipid α-galactosylceramide (αGC) to wild type mice considerably enhances TH1 immune responses in an NKT- and CD1d-dependent manner. In this work we sought to study the in situ role of NKT in alloreactivity. For this purpose, allogeneic mixed lymphocyte reactions (MLR) were performed using 3H incorporation assays. Purified, negatively selected CD3+ cells and irradiated allogeneic peripheral blood mononuclear cells from normal individuals were used as responders and stimulators respectively. After rigorous NKT cell (as identified by staining with the anti-TCRVα24 and -Vβ11 mAbs specific to the NKT TCR α and β chains) depletion by flow sorting we compared MLR reactivity in the presence and absence of NKT. In a series of MLR that included a panel of 4 different responders and 3–5 stimulators, depletion of NKT profoundly suppressed the proliferation by 68.5%±16.9 compared to baseline (i.e., NKT-replete MLR). This suppressive effect was mirrored by a reduction (45.3%±8) of IFNγ secretion in the supernatants of the NKT-depleted MLR compared to baseline. IL-4, IL-10 and TGFβ were not detected in either NKT replete or NKT depleted MLR. When freshly flow-sorted NKT cells were placed against the allogeneic stimulators they did not proliferate indicating that the decrease in the proliferation after NKT cell depletion is due to a decrease in the proliferation of the alloreactive T cells. Taken together, these findings indicate that NKT cells positively regulate the alloresponse, a TH1-driven immune response. Consistent with this, in MLR performed in the presence of αGC (100ng/ml), proliferation was significantly enhanced as compared to baseline MLR (i.e., performed in the presence of the αGC diluent). In a panel of 5 responders against a panel of 3–5 stimulators treatment of the MLR with αGC resulted in a 43.2%±15.2 increase in proliferation. In accordance with the proliferation data, IGNγ production was significantly increased (mean of 53%), whereas IL-4 was undetectable under both conditions. Furthermore, the enhancing effect of αGC was NKT-dependent, as in NKT-depleted MLR proliferation was equally suppressed in the presence of αGC and its diluent (82.6±6.8 vs 82±8.5 respectively, n=3).In summary, we have demonstrated that NKT cells exert an enhancing effect on the alloreactive response and NKT cell depletion effectively suppresses in vitro alloreactivity. These findings set the scene for exploring on one hand the potential for reducing the risk of severe aGVHD by using NKT depletion in allogeneic haemopoietic stem cell transplantation and on the other hand for exploring the adoptive transfer of purified NKT cells to improve immune reconstitution post transplant. In either case, the rapid, accurate identification and physical isolation of the NKT cells is possible either with the use of mAbs highly specific for the TCR of the NKT cells or with the use of the CD1d/α GC tetramer.