Several D-amino acids have been observed in saliva, but their origin and function remain to be clarified. In the present study, large amounts of D-aspartate and small amounts of D-serine and D-alanine were detected in all three major salivary glands in rat. No other D-enantiomers were detected. Protein expression of D-amino acid oxidase and D-aspartate oxidase, the enzymes responsible for the oxidative deamination of neutral and dicarboxylic D-amino acids, respectively, and that of serine racemase, the enzyme converts L-serine to D-serine, were detected in all three major salivary glands in rat. The N-methyl-D-aspartate (NMDA) receptor subunit proteins NR1 and NR2D, but not NR2A, NR2B, or NR2C, were detected in all the three major salivary glands. Perfusion of D-serine with L-glutamate through rat submandibular artery increased salivary secretion during parasympathetic nerve stimulation in a D-serine dose-dependent manner. In vivo microdialysis applied to submandibular glands revealed that perfusion of L-glutamate with D-serine through the microdialysis probe increased acetylcholine contents in interstitial fluids in the glands of anesthetized rats as compared to that with L-glutamate alone in an NMDA receptor antagonist-sensitive manner. The present study suggests that D-amino acids play a physiological role in salivary glands.