The clearance of Ca2+ from nerve terminals is critical for determining the build-up of residual Ca2+ after repetitive presynaptic activity. We found previously that K+-dependent Na+/Ca2+ exchangers (NCKXs) show polarized distributions in axon terminals of supraoptic magnocellular neurons and play a major role in Ca2+ clearance. The role of NCKXs in presynaptic terminals, however, has not been studied. We investigated the contribution of NCKX in conjunction with other Ca2+ clearance mechanisms at the calyx of Held by analyzing the decay of Ca2+ transients evoked by depolarizing pulses. Inhibition of Na+/Ca2+ exchange by replacing external Na+ with Li+ decreased the Ca2+ decay rate by 68%. Selective inhibition of NCKX by replacing internal K+ with TEA+ (tetraethylammonium) or Li+ decreased the Ca2+ decay rate by 42%, and the additional inhibition of the K+-independent form of Na+/Ca2+ exchanger (NCX) by reducing external [Na+] caused an additional decrease by 26%. Inhibition of plasma membrane Ca2+-ATPase (PMCA) decreased the Ca2+ decay rate by 23%, whereas inhibition of SERCA (smooth endoplasmic reticulum Ca2+-ATPase) had no effect. The contribution of mitochondria was negligible for small Ca2+ transients but became apparent at [Ca2+]i > 2.5 microM, when Na+/Ca2+ exchange became saturated. Mitochondrial contribution was also observed when the duration of Ca2+ transients was prolonged by inhibiting Na+/Ca2+ exchangers or by increasing Ca2+ buffers. These results suggest that, in response to small Ca2+ transients (<2 microM), Ca2+ loads are cleared from the calyx of Held by NCKX (42%), NCX (26%), and PMCA (23%), and that mitochondria participate when the Ca2+ load is larger or prolonged.