K-Cl cotransporters (KCCs) play significant roles in epithelial transport and cell volume regulation. Four KCC isoforms (KCC1-4) have been so far reported. We examined whether the KCC isoforms are expressed in the stomach. Western blot analysis showed that KCC3a and KCC4 were expressed in isolated gastric mucosa of rats and mice. Immunohistochemistry in the isolated gastric mucosa showed that KCC3a was expressed in the basolateral membrane and KCC4 was expressed in the apical membrane of the gastric parietal cells. KCC4 protein was found in human gastric tubulovesicles. Interestingly, KCC3a and KCC4 were abundantly expressed in the parietal cells located at luminal segment of the gland. Because the luminal segment parietal cells are much more active in HCl secretion than those of the basal segment, KCC3a and KCC4 may be involved in the mechanism of HCl secretion. In the LLC-PK1 cells stably expressing KCC3a (Tet-on system), ouabain-sensitive Na,K-ATPase activity was significantly higher than that of control cells. The expression level of Na,K-ATPase in the KCC3a-expressing cells was not significantly different from that in the control cells. EP level of Na,K-ATPase in the KCC3a-expressing cells was significantly higher than that in the control cells. KCC3a and Na,K-ATPase were present in the same lipid rafts. KCC3a may regulate gastric acid secretion cooperating with Na,K-ATPase in the basolateral membrane of parietal cells. [J Physiol Sci. 2007;57 Suppl:S77]