Lipopolysaccharide (LPS), a glycolipid found in the outer membrane of Gramnegative bacteria, exerts a variety of biological effects mainly acting on immune cells. A previous study from our group reveal the ability of the endotoxin to exert a direct action on the thyroid cell up-regulating the TSH-stimulated Thyroglobulin gene expression. We previously reported that LPS is also able to stimulate the TSHinduced iodide uptake, a key step in thyroid hormonogenesis, by increasing NIS expression. It has been well characterized in immune cells that LPS acts through Tolllike receptors (TLRs), specifically TLR4. These receptors are a family of proteins related to the Interleukin-1 receptor, with many ligands and able to induce multiple signal transduction pathways. Binding of LPS to TLR4 is complex, other molecules are required to induce the LPS signal. These molecules include CD14, MD-2, and LBP. The present study aimed to elucidate the mechanism by which LPS is recognized in the thyroid cell and to extend our findings on LPS effect on thyroid function. We used a highly differentiated and characterized rat thyroid cell line FRTL5, which has the advantage of being a pure cell preparation with no contaminating cells that could potentially influence the LPS response. We used different approaches to study the expression of TLR4 receptor and accessories molecules, such as RT-PCR and Western blot in membrane fractions. We also studied localization and colocalization by Flow Cytometry, Surface Biotinylation and Confocal Microscopy. FITC-LPS was used to analyze binding by Flow Cytometry. To obtain insight into TLR4 functionality, we performed treatments with different compounds to evaluate specificity and characterize the involvement of TLR4 in the effect of LPS on iodide uptake, NIS protein and mRNA expression. We used Lipid A and Polymyxin as LPS agonist and antagonist respectively, TLR4 specific blocking antibody. We demonstrated, for the first time, that the LPS receptor TLR4, and the molecules CD14 and MD-2, are expressed at the plasma membrane in the thyroid cell and are involved in the LPS recognition. We also evidenced that the TLR4-induced signaling is clearly involved in LPS effect on thyroid function.