The enteric nervous system (ENS) and a glutamate receptor (GluR), N-methyl-D-aspartate receptor (NMDAR), participate in gastric acid secretion (GAS) regulation. NMDARs are localized in different stomach cells; however, knowledge of NMDAR expression and function in the ENS is limited. In the present study, we clarified the types of stomach cells that express the NMDARs that are involved in GAS regulation. The pharmacological method of isolated stomach perfusion by Ghosh and Shild combined with direct mapping of NMDARs by fluorescence microscopy in the rat stomach was employed. By immunofluorescence labeling with an anti-NMDA-NR1 antibody, NMDARs were found to be highly expressed in nerve cells of the submucosal and myenteric plexuses in the stomach. The exact localization of the NMDARs relevant to GAS and its mechanism of action were determined by stimulating different receptors of neuronal and stomach cells using specific secretagogues for NMDA and by selectively blocking those receptors. NMDARs relevant to GAS stimulation are mainly localized in cholinergic interneurons; however, all of the nerve cells of the submucosal ganglia are involved in the stimulating process. In addition, the NMDARs in parietal cells are involved in gastric acid inhibition via influencing H2-histamine receptors.