The NHE4 Na+/H+ exchanger is abundantly expressed on the basolateral membrane of gastric parietal cells. To test the hypothesis that it is required for normal acid secretion, NHE4-null mutant (NHE4-/-) mice were prepared by targeted disruption of the NHE4 (Slc9a4) gene. NHE4-/- mice survived and appeared outwardly normal. Analysis of stomach contents revealed that NHE4-/- mice were hypochlorhydric. The reduction in acid secretion was similar in 18-day-old, 9-week-old, and 6-month-old mice, indicating that the hypochlorhydria phenotype did not progress over time, as was observed in mice lacking the NHE2 Na+/H+ exchanger. Histological abnormalities were observed in the gastric mucosa of 9-week-old NHE4-/- mice, including sharply reduced numbers of parietal cells, a loss of mature chief cells, increased numbers of mucous and undifferentiated cells, and an increase in the number of necrotic and apoptotic cells. NHE4-/- parietal cells exhibited limited development of canalicular membranes and a virtual absence of tubulovesicles, and some of the microvilli had centrally bundled actin. We conclude that NHE4, which may normally be coupled with the AE2 Cl-/HCO3- exchanger, is important for normal levels of gastric acid secretion, gastric epithelial cell differentiation, and development of secretory canalicular and tubulovesicular membranes.