Abstract  NKCC2, an isoform of Na+–K+–2Cl− cotransporter, is principally present in the kidney and plays a critical role in salt reabsorption. Expression of NKCC2 has been found in the apical membrane of intestinal epithelial cells in a number of marine fish, however, details for expression in the mammalian gastrointestinal tract are lacking. RT‐PCR, Western blotting and immunohistochemistry were used to study the expression and localization of NKCC2 in the rat gastrointestinal tract. We found that mRNA transcripts, protein and immunoreactivity (IR) for NKCC2 were expressed in the stomach, small and large intestine of adult rats. NKCC2 IR was localized to the base of the gastric glands, intestinal epithelia, myenteric and submucosal plexuses. NKCC2 IR was expressed strongly in the apical membranes and weakly in the basolateral membranes of intestinal epithelial cells. In the enteric nervous system, NKCC2 IR was widely distributed and localized to enteric neurons with cholinergic, calretinin and nitrergic neuronal immunochemical codes in the myenteric plexus. It was localized to non‐cholinergic secretomotor neurons in the submucosal plexus. In conclusion, this study for the first time clearly detected the expression of NKCC2 in the gastrointestinal tract of a mammalian species. Expression of NKCC2 in gastrointestinal epithelial cells suggested that this cation chloride cotransporter might be involved in gastrointestinal ion transport. Expression of NKCC2 in enteric neurons might contribute to the accumulation of Cl− and a more depolarized ECl− in enteric neurons.