OBJECTIVE: NKT cells promote atherogenesis, but the subtypes responsible have not been identified. We investigated 2 major NKT cell subtypes (CD4+ and DN NKT) in ApoE-/- mice rendered NKT cell-deficient by day-3 neonatal thymectomy (3dTx). METHODS AND RESULTS: Atherosclerosis development was studied in thymectomized ApoE-/- mice fed a high-fat diet with/without adoptively transferred NKT cells. We demonstrate NKT cell deficiency in thymectomized mice and markedly smaller atherosclerotic lesions. The reduction in lesion size was reversed by adoptive transfer of liver-derived NKT cells. Adoptive transfer of CD4+, but not DN NKT cells, into 3dTx ApoE-/- mice increased lesion size 2.5-fold. The differential effects were not attributable to differences in homing to developing atherosclerotic lesions. DN NKT cells expressed at least 3-fold higher levels of inhibitory Ly49 receptors (Ly49A, Ly49C/I, and Ly49G2) than CD4+ NKT cells, and lesions expressed large amounts of their MHC class I ligand. In vitro these inhibitory receptors initiated greater effects in DN NKT cells. Culture of each NKT cell subset with TAP-deficient (MHC class I-deficient) dendritic cells and alpha-GalCer led to secretion of similar amounts of proatherogenic cytokines IL-2, IFN-gamma, and TNF but, when cultured with MHC class I-positive dendritic cells, CD4+ NKT cells secreted more of these cytokines. CONCLUSIONS: CD4+ NKT cells are responsible for the proatherogenic activity of NKT cells. Expression of inhibitory Ly49 receptors by the subtypes appears responsible for regulating their secretion of proatherogenic cytokines and their differential proatherogenic effects.