Studies on hippocampal glycine release are extremely rare. We here investigated release from mouse hippocampus glycinergic terminals selectively pre-labelled with [3H]glycine through transporters of the GLYT2 type. Purified synaptosomes were incubated with [3H]glycine in the presence of the GLYT1 blocker NFPS to abolish uptake (∼ 30%) through GLYT1. The non-GLYT1-mediated uptake was entirely sensitive to the GLYT2 blocker Org25543. Depolarization during superfusion with high-K+ (15–50 mmol/L) provoked overflows totally dependent on external Ca2+, whereas in the spinal cord the 35 or 50 mmol/L KCl-evoked overflow (higher than that in hippocampus) was only partly dependent on extraterminal Ca2+. In the hippocampus, the Ca2+-dependent 4-aminopyridine (1 mmol/L)-evoked overflow was five-fold lower than that in spinal cord. The component of the 10 μmol/L veratridine-induced overflow dependent on external Ca2+ was higher in the hippocampus than that in spinal cord, although the total overflow in the hippocampus was only half of that in the spinal cord. Part of the veratridine-evoked hippocampal overflow occurred by GLYT2 reversal and part by bafilomycin A1-sensitive exocytosis dependent on cytosolic Ca2+ generated through the mitochondrial Na+/Ca2+ exchanger. As glycine sites on NMDA receptors are normally not saturated, understanding mechanisms of glycine release should facilitate pharmacological modulation of NMDA receptor function.