The aim of the present study was to investigate the possibility that ductal cells, which preferentially survive and/or proliferate in Sjögren's syndrome (SS) salivary glands of patients with SS, could acquire the functional expression of membrane water channel aquaporin-5 (AQP5). Thus, in this study, we demonstrate that an immortalized normal human salivary gland ductal cell (NS-SV-DC) line, lacking the expression of AQP5, acquires AQP5 gene expression in response to treatment with 5-aza-2'-deoxycytidine (5-Aza-CdR), a DNA demethylating agent. Confocal microscopic analysis revealed the localization of AQP5 expression mainly at the apical and lateral sides of the plasma membrane. The expressed AQP5 protein was functionally active because AQP5 expression resulted in a significant increase in the osmotically directed net fluid rate across monolayers of NS-SV-DC cells. By the analysis of bisulfite sequencing of CpG islands in the AQP5 promoter, hypermethylation within the consensus Sp1-binding sites was commonly observed in parental cell clones, whereas demethylation at the CGs, one in the second consensus Sp1 element and the other outside of the third consensus Sp1 element in the AQP5 promoter, was detected in NS-SV-DC cells after treatment with 5-Aza-CdR. By analyzing the luciferase activity of transfected AQP5 promoter vectors, it became evident that demethylation at the CGs cooperatively functions between these two sites to induce AQP5 expression. Our data, therefore, suggest that treatment of ductal cells with 5-Aza-CdR could result in the expression of the AQP5 gene, thereby leading to increased fluid secretion from ductal cells in SS salivary glands.