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Although the contribution of Ca2+ buffering systems can vary between neuronal types and cellular compartments, it is unknown whether distinct Ca2+ sources within a neuron have different buffers. As individual Ca2+ sources can have separate functions, we propose that each is handled by unique systems. Using Aplysia californica bag cell neurons, which initiate reproduction through an afterdischarge involving multiple Ca2+-dependent processes, we investigated the role of endoplasmic reticulum (ER) and mitochondrial sequestration, as well as extrusion via the plasma membrane Ca2+-ATPase (PMCA) and Na+/Ca2+ exchanger, to the clearance of voltage-gated Ca2+ influx, Ca2+-induced Ca2+-release (CICR), and store-operated Ca2+ influx. Cultured bag cell neurons were filled with the Ca2+ indicator, fura-PE3, to image Ca2+ under whole-cell voltage clamp. A 5 Hz, 1 min train of depolarizing voltage steps elicited voltage-gated Ca2+ influx followed by EGTA-sensitive CICR from the mitochondria. A compartment model of Ca2+ indicated the effect of EGTA on CICR was due to buffering of released mitochondrial Ca2+ rather than uptake competition. Removal of voltage-gated Ca2+ influx was dominated by the mitochondria and PMCA, with no contribution from the Na+/Ca2+ exchanger or sarcoplasmic/endoplasmic Ca2+-ATPase (SERCA). In contrast, CICR recovery was slowed by eliminating the Na+/Ca2+ exchanger and PMCA. Last, store-operated influx, evoked by ER depletion, was removed by the SERCA and depended on the mitochondrial membrane potential. Our results demonstrate that distinct buffering systems are dedicated to particular Ca2+ sources. In general, this may represent a means to differentially regulate Ca2+-dependent processes, and for Aplysia, influence how reproductive behavior is triggered. [ABSTRACT FROM AUTHOR]