Massive amounts of Ca(2+) can accumulate in mitochondria, owing to its complexation with matrix phosphate. Under conditions in which the mitochondrial uniporter is the foremost pathway for Ca(2+) efflux, the release of sequestered Ca(2+) by protonophoric uncouplers is invariably demonstrated. This has been recently ascribed to matrix acidification, which results in the dissociation of the Ca(2+)-phosphate complex. In the present study, we compared the effect of stepwise depolarization on Ca(2+) release induced by either the complex III inhibitor stigmatellin or an uncoupler in energized Ca(2+)-loaded rat liver mitochondria in the presence of phosphate, at extramitochondrial pH (pH(o)) 6.8 and pH(o) 7.8. Both poisons were examined in the presence and absence of oligomycin. Prior to Ca(2+) loading, mitochondria were allowed to phosphorylate 0.5 mm ADP. Opening of the permeability transition pore was additionally hampered by cyclosporin A, and was monitored by changes in light scattering. Na(+) was excluded from the medium, preventing Na(+)/Ca(2+) exchange. At both pH(o) values, Delta pH was in the range 0.11-0.15. Complete depolarization by uncoupling with or without oligomycin resulted in an approximately pH 0.05 acidic shift, but there was none in the case of stigmatellin plus oligomycin. At pH(o) 6.8 and in the presence of oligomycin, the uncoupler-induced Ca(2+) release started in the -80 to -50 mV range, whereas in the absence of oligomycin, the release occurred at approximately -15 mV. Stigmatellin induced minor Ca(2+) release only in the presence of oligomycin, starting at approximately -4 mV. At pH(o) 7.8, the uncoupler-induced Ca(2+) release started at approximately -11 mV, irrespective of the presence or absence of oligomycin. Unexpectedly, at this alkaline pH and in the presence of oligomycin, stigmatellin induced substantial Ca(2+) release, starting at approximately -10 mV. From the above findings, we conclude that matrix acidification cannot be the sole explanation for uncoupler-induced Ca(2+) release from mitochondria.