Abstract: We compared the activity of free d-Ser on the potentiation of cloned NMDA receptors with that of Gly by using a Xenopus oocyte expression system. The extracellular concentration of free d-Ser and Gly was further studied by means of microdialysis. The ED50 values of d-Ser were three to four times lower than those of Gly in any combination of ε1, ε2, ε3, or ε4 and ζ1. Site-directed mutagenesis of ζ1 subunits revealed that some aromatic residues necessary for the action of Gly affected the ED50 value of d-Ser. This result showed that the residues play crucial roles in the action of d-Ser. In vivo microdialysis of rodent brain revealed that the extracellular concentration of free d-Ser in the frontal cortex (6.5 µM) was high enough to saturate the Gly site on the NMDA receptor, but that in the cerebellum was not. These findings suggest that d-Ser is a candidate of the endogenous potentiator of the NMDA receptor in the rodent frontal cortex.