In salivary glands, primary saliva is produced by acini and is modified by the reabsorption and secretion of ions in the ducts. Thus, the permeability of intercellular junctions in the ducts is considered to be lower than in the acini. We have examined the relationship between the expressed claudin isotypes and the barrier functions of tight junctions in a submandibular gland epithelial cell line, SMIE. SMIE cells were originally derived from rat submandibular duct cells, but their barrier functions are not as efficient as those of Madin-Darby canine kidney cells. Large molecules, such as 70-kDa dextran, diffuse across the monolayers, although E-cadherin and occludin, adherens junction and tight junction proteins, respectively, are expressed in SMIE cells. Claudin-3 protein has also been detected, but the expression level of claudin-3 mRNA is much lower than in the original submandibular glands. Other claudins including claudin-4 (originally expressed in the duct cells) have not been detected. Because of the limited expression of claudins, SMIE cells are suitable for studying the role(s) of claudins. To examine the function of claudin-4 in submandibular glands, we have overexpressed green fluorescence protein (GFP)-fused claudin-4 in SMIE cells. Cells that express GFP-fused claudin-4 have a higher transepithelial electrical resistance and a lower permeability of 70-kDa dextran, although the expression levels of occludin and claudin-3 are hardly affected. Therefore, claudin-4 plays a role in the regulation of the barrier function of tight junctions in submandibular glands.