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 ~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‐adeno‐sylmethionine‐dependent enzyme, which we tentatively denominated as NMDA synthase.—D‧Aniello, A., Di Fiore, M. M., Fisher, G. H., Milone, A., Seleni, A., D'Aniello, S., Perna, A. F., Ingrosso, D. Occurrence of D‐aspartic acid and N‐methyl‐D‐aspartic acid in rat neuroendocrine tissues and their role in the modulation of luteinizing hormone and growth hormone release. FASEB J. 14, 699–714 (2000)