Toll-like receptors (TLR) play an essential role in the activation of both innate and adaptive immune responses. Salivary gland epithelial cells (SGEC) may participate in the development of glandular inflammatory reactions that characterize primary Sjögren's syndrome (pSS). In this study we sought to assess the expression and function of several TLR molecules in cultured non-neoplastic SGEC obtained from pSS patients and disease controls. Long-term cultured non-neoplastic SGEC derived from pSS patients (SS-SGEC) and disease controls (control-SGEC), as well as the monocytic cell line THP-1 (positive control cell line), were examined by reverse transcription–polymerase chain reaction (RT–PCR) analysis and quantitative real-time PCR for mRNA expression of TLR1, −2, −3 and −4 molecules. TLR function was assessed by the induction of the expression (flow cytometry) of the immunoregulatory molecules CD54/intercellular adhesion molecule-1 (ICAM-1), CD40, CD86/B7·2, major histocompatibility complex (MHC) class I and MHC class II following treatment with the TLR ligands: Staphylococcus aureus peptidoglycan (TLR2), the synthetic dsRNA analogue polyinosinic:cytidylic acid (TLR3) and Escherichia coli lipopolysaccharide (TLR4). SGEC were found to express functional TLR2, −3 and −4 molecules, as attested by dose-dependent up-regulation of surface ICAM-1, CD40 and MHC-I expression (as well as of reciprocal TLR mRNA) following treatment with the respective TLR-ligands. SS-SGEC lines displayed significantly higher constitutive expression of TLR1 (P = 0·0027), TLR2 (P = 0·01) and TLR4 (P = 0·03) mRNA compared to control-SGEC. This study demonstrates that cultured SGEC express functional TLR molecules; the high constitutive TLR expression by SS-SGEC is probably suggestive of the intrinsic activation of epithelial cells in pSS and further supports the role of this type of tissue in pathogenesis of the disorder.