Pinealocytes, endocrine cells that synthesize and secrete melatonin, possess a large number of synaptic-like microvesicles (MVs) containing the L-glutamate transporter (Moriyama, Y., and Yamamoto, A. (1995) FEBS Lett., 367, 233-236). In this study, the L-glutamate transporter in MVs isolated from bovine pineal glands was characterized as to its driving force, requirement of anions, and substrate specificity. Upon the addition of ATP, the MVs accumulated L-glutamate. The uptake was significantly dependent on the extravesicular Cl ؊ concentration, being negligible in the absence of Cl ؊ and maximum at 2-5 mM and decreasing gradually at 20 -100 mM. The membrane potential (inside positive) was maximum at 0 -10 mM Cl ؊ and then decreased gradually depending on the Cl ؊ concentration, whereas a pH gradient was practically absent without Cl ؊ and increased gradually up to 100 mM Cl ؊ . Ammonium acetate or nigericin plus K ؉ , a dissipator of a pH gradient, had little effect on or was slightly stimulatory toward the uptake, whereas valinomycin plus K ؉ inhibited both formation of the membrane potential and the glutamate uptake to similar extents. The ATP-and Cl ؊ -dependent glutamate uptake was inhibited by fluoride, iodide, or thiocyanate, without vacuolar H ؉ -ATPase being affected. An anion channel blocker, 4,4-diisothiocyanatostilbene-2,2-disulfonic acid, similarly inhibited the glutamate uptake in a Cl ؊ protectable manner. Furthermore, ATP-and glutamate-dependent acidification of MVs was observed when 4 mM Cl ؊ was present. Among more than 50 kinds of glutamate analogues tested, only a few compounds, including 1-aminocyclohexane-trans-1,3-dicarboxylic acid, caused similar acidification. A good correlation was observed between the acidification and the inhibition of glutamate uptake by glutamate analogues. These results indicated that 1) the major driving force of the glutamate uptake is the membrane potential, 2) Cl ؊ regulates the glutamate uptake, probably via anion-binding site(s) on the transporter, and 3) the transporter shows strict substrate specificity. Hence, the overall properties of the vesicular glutamate transporter in the MVs well matched those of the synaptic vesicle glutamate transporter. We concluded that the vesicular glutamate transporter, being similar if not identical to the neuronal counterpart, operates in endocrine cells.Pinealocytes are parenchymal endocrine cells of pineal glands that synthesize and secrete melatonin into the blood (1-3). At least two kinds of secretory vesicle-like organelles have been identified in pinealocytes: dense core granules, which are speculated to be involved in the storage and secretion of melatonin and some neuropeptides such as arginine vasotocin (3, 4), and a large number of synaptic-like microvesicles (MVs) 1 containing synaptophysin (5). Although histochemical evidence indicated that MVs are distinct from neuronal synaptic vesicles in their lack of synapsin I, the possible participation of MVs in some secretory pathways in pinealocytes was also speculated...