Epithelial cells appear to play an important role in the initiation and maintenance of autoimmune lesions in the salivary glands of patients with Sjögren's syndrome. Therefore, the detailed study of immunological function of salivary gland epithelial cells (SGEC) may provide useful information for the understanding of Sjögren's syndrome pathogenesis. In this report we aimed to formulate a protocol for the establishment of human non-neoplastic SGEC lines as a tool for the study of the physiology and pathophysiology of these cells. Pointing towards a practical approach, we sought to establish SGEC lines from quite a limited amount of biopsy tissue obtained during the diagnostic evaluation of patients. Herein, the favorable conditions for the long-term maintenance of human non-neoplastic SGEC lines are presented and involve the successive application of a serum-containing and a serum-free culture medium, supplemented with essential epithelial growth factors. This protocol has been found reliable and convenient, as attested by the reproducible establishment of non-neoplastic SGEC lines. The analysis of SGEC phenotypic features, as well as a coculture system for the study of interactions between epithelial cells and lymphocytes, are also described. Such techniques may provide valuable means for the functional and molecular investigation of human SGEC and particularly for the study of Sjögren's syndrome and other disorders of glandular epithelia.