Objective-Atherosclerosis encompasses a conspicuously maladaptive inflammatory response that might involve innate immunity. Here, we compared the role of Toll-like receptor 4 (TLR4) with that of TLR2 in intimal foam cell accumulation and inflammation in apolipoprotein E (ApoE) knockout (KO) mice in vivo and determined potential mechanisms of upstream activation and downstream action. Methods and Results-We measured lipid accumulation and gene expression in the lesion-prone lesser curvature of the aortic arch. TLR4 deficiency reduced intimal lipid by Ϸ75% in ApoE KO mice, despite unaltered total serum cholesterol and triglyceride levels, whereas TLR2 deficiency reduced it by Ϸ45%. TLR4 deficiency prevented the increased interleukin-1␣ (IL-1␣) and monocyte chemoattractant protein-1 mRNA levels seen within lesional tissue, and it also lowered serum IL-1␣ levels. Smooth muscle cells (SMC) were present within the intima of the lesser curvature of the aortic arch at this early lesion stage, and they enveloped and permeated nascent lesions, which consisted of focal clusters of foam cells. Cholesterol enrichment of SMC in vitro stimulated acyl-coenzyme A:cholesterol acyltransferase-1 mRNA expression, cytoplasmic cholesterol ester accumulation, and monocyte chemoattractant protein-1 mRNA and protein expression in a TLR4-dependent manner. A therogenesis involves a strikingly maladaptive inflammatory response, initially to retained and modified lipoproteins 1 and later to apoptotic or necrotic cell debris that accumulates within the arterial wall. 2 Toll-like receptors (TLR) elicit innate immune responses and inflammation when activated by either exogenous microbial products or endogenous molecules with similar structural features, and they are candidate mediators of atherogenic inflammation. TLR2 and TLR4 are expressed in arterial lesions of mice and humans, 3,4 and exogenous microbial TLR2 and TLR4 agonist ligands promote atherosclerosis in hypercholesterolemic mice, 5,6 implicating these receptors as candidate atherogenic mediators. In gene knockout (KO) mouse models, TLR2 deficiency reduced early atherogenic events and later aortic lesion development. 7,8 In contrast, deficiency of TLR4, the bacterial lipopolysaccharide (LPS) receptor, produced modest effects 9 or no effects 10 on aortic lesion burden in studies limited to advanced disease stages. However, prolonged and severe hypercholesterolemia may generate distinct modified lipids or proteins that promote inflammation and atherogenesis via distinct, TLR-independent pathways. Therefore, here we tested the hypothesis that TLR4 promotes atherogenesis during early-stage disease, using a mouse model of shorterterm and less severe hypercholesterolemia, and compared its influence to that of TLR2. Conclusion-TLR4TLR signaling strongly activates proinflammatory genes in multiple cell types found within early atherosclerotic lesions, but little is known about how this promotes atherogenesis. Such atherogenic TLR signaling may not be restricted to hematopoietic cells, beca...