Toll-like receptors (TLRs) mediate the cellular response to conserved molecular patterns shared by microorganisms. We report that TLR-2 on human NK cells is upregulated and stimulated by Leishmania major lipophosphoglycan (LPG), a phosphoglycan belonging to a family of unique Leishmania glycoconjugates. We found that purified L. major LPG upregulates both mRNA and the membrane expression of TLR-2 in NK cells. Additionally, IFN-gamma and TNF-alpha production and nuclear translocation of NF-kappaB was enhanced. The activation effect was more intense with LPG purified from infectious metacyclic parasites than from noninfectious procyclic Leishmania. Since the difference between the molecules derived from these two stages of the parasite growth cycle lies exclusively in the number of phosphosaccharide repeat domains and in the composition of glycan side chains that branch off these domains, we propose that TLR-2 possibly distinguishes between phosphorylated glycan repeats on LPG molecules. The effect of LPG on cytokine production and on membrane expression of TLR-2 could be blocked with F(ab')2 fragments of the mAb against LPG (WIC 79.3). Confocal microscopy demonstrated the co-localization of LPG and TLR-2 on the NK cell membrane. Binding of LPG to TLR-2 in NK cells was demonstrated by immunoprecipitations done with anti-TLR-2 and anti-LPG mAb followed by immunoblotting with anti-LPG and anti-TLR-2, respectively. Both antibodies recognized the immune complexes. These results suggest that NK cells are capable of recognition of, and activation by, Leishmania LPG through TLR-2, enabling them to participate autonomously in the innate immune system and thereby increasing the effective destruction of the parasite.