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 minutes, 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⁺⁺ which enters the presynaptic terminals mostly through voltage-gated Ca⁺⁺ channels but also, to a much lesser degree, through a pathway independent of these channels or reverse mode of the plasma membrane Na⁺/Ca⁺⁺ 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⁺⁺ uptake or mitochondrial Na⁺/Ca⁺⁺ exchanger. mEPSCs amplitude is also potentiated by HFS which requires extracellular Ca⁺⁺. In conclusion, induction of mEPSC-STP is redundantly mediated by presynaptic N-, P/Q- and L-type Ca⁺⁺ channels while the second phase depends on mitochondrial Ca⁺⁺ 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.