The gliotransmitter D-serine is released upon (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate and metabotropic glutamate receptor stimulation, but the mechanisms involved are unknown. Here, by using a highly sensitive bioassay to continuously monitor extracellular D-serine levels, we have investigated the pathways used in its release. We reveal that D-serine release is inhibited by removal of extracellular calcium and augmented by increasing extracellular calcium or after treatment with the Ca(2+) ionophore A23187. Furthermore, release of the amino acid is considerably reduced after depletion of thapsigargin-sensitive intracellular Ca(2+) stores or chelation of intracellular Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate-acetoxymethyl ester. Interestingly, D-serine release also was markedly reduced by concanamycin A, a vacuolar-type H(+)-ATPase inhibitor, indicating a role for the vesicular proton gradient in the transmitter storage/release. In addition, agonist-evoked D-serine release was sensitive to tetanus neurotoxin. Finally, immunocytochemical and sucrose density gradient analysis revealed that a large fraction of D-serine colocalized with synaptobrevin/VAMP2, suggesting that it is stored in VAMP2-bearing vesicles. In summary, our study reveals the cellular mechanisms subserving D-serine release and highlights the importance of the glial cell exocytotic pathway in influencing CNS levels of extracellular D-serine.