The rate and the total amount of Ca2+ elevation in the presynaptic terminals of bullfrog sympathetic ganglia depend on the firing frequency of the terminals. Carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, was used for testing whether mitochondrial Ca2+ uptake is one of the mechanisms that underlie this frequency dependence. Fura-2 fluorimetry was used for measurement of intraterminal Ca2+. When stimulations of different durations (30 and 1.5 s) and frequencies (4 and 20 Hz) evoked Ca2+ transients with similar peak amplitudes (264 +/- 22 nM vs. 251 +/- 18 nM, means +/- SE), CCCP augmented the responses to the 4-Hz stimulation 8.9 times more strongly than it did the responses to the 20-Hz stimulation (249.7 +/- 81.5% vs. 25.3 +/- 10.2%). When stimulations delivered at the two frequencies had the same durations (1.5, 3, 6, 10, 20, and 30 s), CCCP enlarged the responses to the 4-Hz stimulations up to 4.2 times more than it did the responses to the 20-Hz stimulations. When the same number of stimuli (120) was delivered at the two frequencies, the effects of CCCP on the responses evoked by the 4-Hz train were again 6.8 times stronger than its effects on the responses to the 20-Hz stimulation. Therefore neither the peak amplitudes of the responses nor the durations of the stimulations dictated the extent to which the mitochondria modulated the peak [Ca2+]i. Instead, the extent of the modulation was governed by the frequency of stimulation. Specifically, the less frequent the Ca2+ influx, the stronger the mitochondrial modulation. Also, during nerve firing Ca2+ release from the ryanodine-sensitive store had a higher potential to influence the [Ca2+]i transients than did Ca2+ removal by the mitochondria for the first 6 s of the responses. On cessation of stimulation, CCCP reduced the initial rapid rate of Ca2+ decay. Thus uptake by the mitochondria was an important mechanism for Ca2+ removal after repetitive firing at the presynaptic terminals.