Toll-like receptors (TLRs) initiate and maintain host defenses and inflammation, and directly contribute to diseases such as atherosclerosis. It is not completely understood in what cell types proatherogenic TLR-induced signaling arises and, particularly, there is uncertainty regarding the potential functional role of TLR2 in endothelial cells (ECs). We determined TLR2 and TLR4 gene expression in four different human and two different murine primary ECs using gene array analysis, RT-PCR, and flow cytometry and confirmed these data by functional studies by stimulating ECs with the corresponding TLR ligands. TLR4 was expressed in all human and murine ECs and these cells responded to stimulation with LPS. Faint expression of TLR2 was observed in human ECs, whereas murine ECs express considerable amounts of TLR2 mRNA. Human ECs failed to respond to TLR2 ligands while murine ECs responded to TLR2 ligands. Furthermore, in murine ECs, TLR2 was located on the cell surface while in human ECs, TLR2 was sequestered in intracellular compartments. After IFN-gamma or IL-1beta stimulation, TLR2 translocated to the cell surface of human ECs. In conclusion, TLR2 is expressed intracellularly in human ECs and, therefore, TLR2 ligands are inaccessible to the receptor. Murine ECs express membrane TLR2 and respond to TLR2 ligands, but human ECs normally will not respond unless they are first primed with inflammatory stimulation, which appears to trigger translocation of TLR2 to the cell surface.