Using two specific and sensitive fluorometric/HPLC methods and a GC-MS method, alone and in combination with D-aspartate oxidase, we have demonstrated for the first time that N-methyl-D-aspartate (NMDA), in addition to D-aspartate (D-Asp), is endogenously present as a natural molecule in rat nervous system and endocrine glands. Both of these amino acids are mostly concentrated at nmol/g levels in the adenohypophysis, hypothalamus, brain, and testis. The adenohypophysis maximally showed the ability to accumulate D-Asp when the latter is exogenously administered. In vivo experiments, consisting of the i.p. injection of D-Asp, showed that D-Asp induced both growth hormone and luteinizing hormone (LH) release. However, in vitro experiments showed that D-Asp was able to induce LH release from adenohypophysis only when this gland was co-incubated with the hypothalamus. This is because D-Asp also induces the release of GnRH from the hypothalamus, which in turn is directly responsible for the D-Asp-induced LH secretion from the pituitary gland. Compared to D-Asp, NMDA elicits its hormone release action at concentrations approximately 100-fold lower than D-Asp. D-AP5, a specific NMDA receptor antagonist, inhibited D-Asp and NMDA hormonal activity, demonstrating that these actions are mediated by NMDA receptors. NMDA is biosynthesized from D-Asp by an S-adenosylmethionine-dependent enzyme, which we tentatively denominated as NMDA synthase.