To study the role of mitochondrial Ca 2+ clearance in stimulated cells, changes in free Ca 2+ concentration in the cytosol, [Ca 2+] c and that in mitochondria, [Ca 2+] m along with secretory responses were observed using chromaffin cells co-loaded with Fura-2 and Rhod-2 in the perfused rat adrenal medulla. When the cells were stimulated with 40 mM K + in the perfusate, the duration of [Ca 2+] m response markedly increased with prolongation of the stimulation period, exhibiting a mean half-decay time of 21 min with 30 s stimulation, whereas its amplitude was not altered with stimulations of 10–30 s. A computer simulation analysis showed that such a mode of [Ca 2+] m response can be produced if excess Ca 2+ taken up by mitochondria precipitates as calcium phosphate (Pi) salt. In the presence of 5 μM rotenone plus 10 μM oligomycin, a decrease in the duration of [Ca 2+] m response and a slight but significant increase (24%) in the secretory response to 30 s stimulation with 40 mM K + were observed. Simulation analyses suggested that this effect of rotenone may be due to reduction in mitochondrial Ca 2+ uptake induced by rotenone-elicited partial depolarization of the mitochondrial membrane potential. In chromaffin cells transsynaptically stimulated through the splanchnic nerve, the intensity of NAD(P)H autofluorescence changed with time courses similar to those of [Ca 2+] m responses. The temporal profiles of those two responses were prolonged in a similar manner by application of an inhibitor of mitochondrial Na +/Ca 2+ exchanger, CGP37157. Thus, due to the unique Ca 2+ buffering mechanism, [Ca 2+] m responses associated with massive mitochondrial Ca 2+ uptake may occur within a limited concentration range in which Ca 2+-sensitive dehydrogenases are activated to control the mitochondrial redox state in stimulated chromaffin cells.