Vesicular glutamate transporter is present in neuronal synaptic vesicles and endocrine synaptic-like microvesicles, and is responsible for vesicular storage of L-glutamate. A brain-specific Na+-dependent inorganic phosphate transporter (BNPI) functions as a vesicular glutamate transporter in synaptic vesicles, and the expression of this BNPI defines the glutamatergic phenotype in the central nervous system (Bellocchio, E. E., et al., (2000) Science, 289, 957-960; Takamori, S., et al., (2000) Nature, 407, 189-194). However, since not all glutamatergic neurons contain BNPI, an additional transporter(s) responsible for vesicular glutamate uptake has been postulated. Here, we report that differentiation-associated Na+-dependent inorganic phosphate cotransporter (DNPI), an isoform of BNPI (Aihara, Y., et al., (2000) J. Neurochem. 74, 2622-2625), also transports L-glutamate at the expense of an electrochemical gradient of protons established by the vacuolar proton pump when expressed in COS7 cells. Molecular biological and immunohistochemical studies have indicated that besides in neuronal cells DNPI is preferentially expressed in mammalian pinealocytes, aTC6 cells, clonal pancreatic a cells, and a cells of Langerhans islets, these cells being proven to secrete L-glutamate through Ca2+-dependent regulated exocytosis followed by its vesicular storage. Pancreatic polypeptide-secreting F cells of Langerhans islets also expressed DNPI. These results constitute evidence that DNPI functions as another vesicular transporter in glutamatergic endocrine cells as well as neurons.