High levels of D-aspartate occur in the brain and endocrine glands, such as pineal, adrenal and pituitary. In the brain, D-aspartate levels are highest in embryonic and early postnatal stages. Notably high levels occur in the early postnatal cortical plate and subventricular zone of the cerebral cortical cultures, implying a role in development. In embryonic neuronal primary culture cells, we detected high levels of endogenous D-aspartate and demonstrated biosynthesis of [14C]D-aspartate using [14C]L-aspartate as precursor. Synthesis of D-aspartate in cell cultures is inhibited by amino-oxyacetic acid, an inhibitor of pyridoxal phosphate-dependent enzymes. In the rat adrenal medulla, D-aspartate is depleted by treatment of the animals with intraperitoneal nicotine injections. In adrenal slices, D-aspartate is released by depolarization with KCl or acetylcholine, implying physiological release by activation of the cholinergic innervation of the adrenal. Our characterization of D-aspartate ontogeny, biosynthesis and depolarization-induced release implies specific physiological roles for this amino acid.