In the present study we tested the responsiveness of human corneal epithelial cells (HCECs) and corneal fibroblasts to lipopolysaccharide (LPS), a TLR4 ligand. Purified P aeruginosa LPS was used to stimulate telomerase-immortalized HCECs (HUCL) and stromal fibroblast (THK) cell lines. Exposure of cells to LPS induced a time-dependent activation of NF-κB in THK but not in HUCL cells, as assessed by an increase in IκB-α phosphorylation and degradation. Concomitant with NF-κB activation, LPS-treated THK cells, but not HUCL cells, produced significantly more cytokines than control untreated cells. A cell surface biotinylation assay revealed that HUCL cells express TLR4 intracellularly whereas TLR5 is expressed on the cell surface. Furthermore, RT-PCR analysis revealed that HUCL and primary HCECs, in contrast to THK cells, do not express MD-2. Thus, our results demonstrate that the LPS unresponsiveness of HCECs might be due to deficient expression of MD2, an essential component for LPS-TLR4 signaling.