D-Aspartate is present in the central nervous system and various endocrine organs, and modulates their neuroendocrine function. In islets of Langerhans, α and β cells contain D-aspartate. Here we show that INS-1E clonal β cells contain the highest amount of D-aspartate. Immunohistochemical analysis with specific antibodies against D-aspartate indicated that D-aspartate is co-localized with insulin. Upon the addition of K+, both D-aspartate and insulin are secreted from the cells in a Ca2+-dependent manner. A Ca2+ ionophore, A23187, also triggers the release of D-aspartate and insulin in the presence of Ca2+. Bafilomycin A1, a specific inhibitor of V-ATPase and V-ATPase-linked secondary transport, inhibits the secretion of D-aspartate. These results support the idea that D-aspartate is present in insulin-containing secretory granules and co-secreted with insulin through exocytosis.