Similar to cell shrinkage, membrane blebbing, DNA fragmentation and phosphatidylserine exposure, intracellular acidification is a hallmark of apoptosis. It has been proposed that mitochondria play a pivotal role in apoptosis-related cytosolic acidification. The present study explored the involvement of mitochondria in the triggering of cytosolic acidification during apoptosis. To this end, we blocked the mitochondrial death pathway by overexpression of the anti-apoptotic protein Bcl-2 and subsequently activated the death receptor pathway by anti-CD95 or TRAIL or the mitochondrial pathway by staurosporine. We show that Bcl-2 but not caspase inhibition prevented staurosporine-induced intracellular acidification. Thus, intracellular acidification in mitochondrial apoptosis is a Bcl-2- inhibitable, but caspase-independent process. In contrast, Bcl-2 delayed, but did not prevent intracellular acidification upon triggering of death receptors. The Na+ /H+ exchanger NHE1 was degraded only to a small extent and at a time point where cytosolic acidification was almost completed. Thus, cytosolic acidification is mitochondrially controlled in response to mitochondria-dependent death stimuli, but involves caspase- dependent mechanisms during death receptor-mediated apoptosis.