The sensitivity of posttetanic potentiation (PTP) to high-frequency stimulation (HFS) steeply decays during the first 2 postnatal weeks. We investigated the underlying mechanisms for the developmental change of PTP induced by HFS (100 Hz, 2 s) at postnatal days 4-6 and 9-11 at the rat calyx of Held synapse. Low-concentration tetraphenylphosphonium (2 μM), an inhibitor of mitochondrial Na(+)/Ca(2+) exchanger, suppressed the amount of posttetanic residual Ca(2+) and PTP to a larger extent at the immature calyx synapse, indicating a developmental reduction of mitochondrial contribution to PTP. The higher amount of mitochondrial Ca(2+) uptake during HFS and consequent posttetanic residual Ca(2+) at the immature calyx of Held was associated with higher peak of HFS-induced Ca(2+) transients, most likely because the mitochondrial Ca(2+) uptake during HFS was supralinearly dependent on the presynaptic resting Ca(2+) level. Probing into the contribution of Na(+)/Ca(2+) exchangers to Ca(2+) clearance, we found a specific upregulation of the K(+)-dependent Na(+)/Ca(2+) exchanger (NCKX) activity in the mature calyx of Held. We conclude that the upregulation of NCKX limits the Ca(2+) buildup and inhibits mitochondrial Ca(2+) uptake during HFS, which in turn results in the reduction of posttetanic residual Ca(2+) and PTP at the mature calyx of Held synapse.