The glutamatergic synapses of the supraoptic nucleus display a unique activity-dependent plasticity characterized by a barrage of tetrodotoxin-resistant miniature EPSCs (mEPSCs) persisting for 5–20 min, causing postsynaptic excitation. We investigated how this short-term synaptic potentiation (STP) induced by a brief high-frequency stimulation (HFS) of afferents was initiated and maintained without lingering presynaptic firing, using in vitro patch-clamp recording on rat brain slices. We found that following the immediate rise in mEPSC frequency, STP decayed with two-exponential functions indicative of two discrete phases. STP depends entirely on extracellular Ca 2+ which enters the presynaptic terminals through voltage-gated Ca 2+ channels but also, to a much lesser degree, through a pathway independent of these channels or reverse mode of the plasma membrane Na + –Ca 2+ exchanger. Initiation of STP is largely mediated by any of the N-, P/Q- or L-type channels, and only a simultaneous application of specific blockers for all these channels attenuates STP. Furthermore, the second phase of STP is curtailed by the inhibition of mitochondrial Ca 2+ uptake or mitochondrial Na + –Ca 2+ exchanger. mEPSCs amplitude is also potentiated by HFS which requires extracellular Ca 2+ . In conclusion, induction of mEPSC-STP is redundantly mediated by presynaptic N-, P/Q- and L-type Ca 2+ channels while the second phase depends on mitochondrial Ca 2+ sequestration and release. Since glutamate influences unique firing patterns that optimize hormone release by supraoptic magnocellular neurons, a prolonged barrage of spontaneous excitatory transmission may aid in the induction of respective firing activities.