The relative contribution of epithelial Sertoli cells in response to bacterial infection of the testis remains poorly characterised, since studies on inflammatory properties of these cells have invariably used unpurified lipopolysaccharide (LPS) preparations contaminated with bacterial lipopeptides. Consequently, isolated rat Sertoli cells were stimulated with either unextracted or phenol re-extracted LPS, and analysed for Toll-like receptor (TLR) 4, TLR2 and inflammatory cytokine gene expression by quantitative reverse transcription polymerase chain reaction (RT-PCR). Expression of TLR4 and its co-receptor protein myeloid differentiation (MD) 2 in Sertoli cells and testicular macrophages were similar, but Sertoli cells displayed low basal or LPS-induced expression of the TLR4 accessory protein, CD14. In Sertoli cells, unextracted LPS produced cytokine responses which were considerably greater in magnitude and duration compared with their response to purified LPS. Sertoli cells also responded to the synthetic lipopeptide, Pam(3)Cys (a TLR2 ligand) with a similar pattern of prolonged gene expression. Sertoli cells were more than 10-fold less sensitive to purified LPS than macrophages, but expressed similar levels of interleukin (IL)-1α and IL-6, and much greater levels of the immunoregulatory cytokine activin A, when maximally stimulated. These data demonstrate that Sertoli cells display differential cytokine responses to bacterial stimuli, mediated by both TLR2 and TLR4, that are distinct from those of testicular macrophages.