Acting through CD14 and TLR4/MD-2, lipopolysaccharide (LPS) triggers strong pro-inflammatory activation of macrophages, which, if not appropriately controlled, may lead to lethal septic shock. Therefore, numerous mechanisms of negative regulation of responses to LPS exist, but whether they include down-regulation of LPS receptors is not clear. We have found that in J774 cells, the clathrin-dependent endocytic pathway enables activation of TRIF-dependent TLR4 signaling within endosomes, but is not associated with the down-regulation of TLR4 or CD14 surface expression. In contrast, lipid raft-dependent endocytosis negatively regulates the basal cell surface expression of LPS receptors and, consequently, responsiveness to LPS. Together with observations that treatments, known to selectively disrupt lipid rafts, do not inhibit LPS-stimulated cytokine production, our results suggest that lipid rafts may serve as sites in which LPS receptors are sorted for endocytosis, rather than being platforms for the assembly of TLR4-centered signaling complexes, as suggested previously.