The expression and function of Toll-like receptor 5 (TLR5) was analysed in human conjunctival epithelial cells (HCjEC).The expression of TLR5 in HCjEC was studied by reverse transcriptase (RT) PCR and flow cytometry. The amount of interleukin (IL) 6 and IL-8 proteins was determined by ELISA. Messenger RNA expression elicited by stimulation with flagellins derived from Pseudomonas aeruginosa, Serratia marcescens, Salmonella typhimurium, and Bacillus subtilis was assayed by quantitative RT-PCR. The localisation of TLR5 protein in human conjunctival epithelium was detected immunohistochemically.HCjEC expressed TLR5-specific mRNA and TLR5 protein. In HCjEC stimulated with flagellins derived from P. aeruginosa and S. marcescens, IL-6 and IL-8 production was increased and IL-6 and IL-8 mRNA was upregulated. Flagellins from S. typhimurium and B. subtilis did not induce the upregulation of these genes and proteins. TLR5 protein was detected on the basolateral but not the apical side of human conjunctival epithelium.Human conjunctival epithelium harbours functional TLR5. Considering the spatially selective basolateral localisation of TLR5 protein, it was postulated that flagellins from ocular pathogenic bacteria induce inflammatory responses when disruption of the epithelial barrier permits their transmigration to the basolateral side but not under healthy physiological conditions on the ocular surface.