Toll-like receptors (TLRs) have recently been identified as fundamental components of the innate immune response to bacterial pathogens. We investigated the role of TLR signaling in immune defense of the mucosal epithelial cells of the lower female genital tract. This site provides first line defense against microbial pathogens while remaining tolerant to a complex biosystem of resident microbiota. Epithelial cells derived from normal human vagina, ectocervix, and endocervix expressed mRNA for TLR1, -2, -3, -5, and -6. However, they failed to express TLR4 as well as MD2, two essential components of the receptor complex for LPS in phagocytes and endothelial cells. Consistent with this, endocervical epithelial cells were unresponsive to protein-free preparations of lipooligosaccharide from Neisseria gonorrhoeae and LPS from Escherichia coli. However, they were capable of responding to whole Gram-negative bacteria and bacterial lysates, as demonstrated by NF-kappaB activation and proinflammatory cytokine production. The presence of soluble CD14, a high-affinity receptor for LPS and other bacterial ligands, enhanced the sensitivity of genital tract epithelial cells to both low and high concentrations of bacteria, suggesting that soluble CD14 can act as a coreceptor for non-TLR4 ligands. These data demonstrate that the response to N. gonorrhoeae and other Gram-negative bacteria at the mucosal surface of the female genital tract occurs in the absence of endotoxin recognition and TLR4-mediated signaling.