Renal tubular epithelial cells (TECs) respond diffusely to local infection, with the release of multiple cytokines, chemokines and other factors that are thought to orchestrate the cellular constituents of the innate immune response. We have investigated whether the Toll-like receptors TLR4 and TLR2, which are present on tubular epithelium and potentially detect a range of bacterial components, co-ordinate this inflammatory response acting through nuclear factor-kappa B (NF-κB). Primary cultures of TECs were grown from C57BL/6, C3H/HeN, C3H/HeJ, TLR2 and TLR4 knock-out mice. Cell monolayers were stimulated with lipopolysaccharide (LPS) and synthetic TLR2 and 4 agonists. The innate immune response was quantified by measurement of the cytokines tumour necrosis factor (TNF)-α and KC (IL-8 homologue) in cell supernatants by enzyme-linked immunosorbent assay. Cultured TECs grown from healthy mice produced the cytokines TNF-α and KC in response to stimulation by LPS and synthetic TLR2 and TLR4 agonists. Cells lacking the respective TLRs had a reduced response to stimulation. The TLR2- and TLR4-mediated response to stimulation was dependent on NF-κB signalling, as shown by curcumin pretreatment of TECs. Finally, apical stimulation of these TLRs elicited basal surface secretion of TNF-α and KC (as well as the reverse), consistent with the biological response in vivo. Our data highlight the potential importance of TLR-dependent mechanisms co-ordinating the innate immune response to upper urinary tract infection.