We have studied the influence of pH on opening of the mitochondrial permeability transition pore (PTP) in both deenergized and energized mitochondria in the presence of Pi. In deenergized mitochondria from rat brain and heart, we observed the expected inhibition of Ca2+-induced PTP opening at increasingly acidic pH values. Unexpectedly, mitochondria energized with either electron transport complex I or complex II substrates displayed the opposite behavior, acidic pH promoting rather than inhibiting PTP opening. We show that the potentiating effect of acidic pH is due to an increased rate of Pi uptake. The data also revealed that brain mitochondria are more heterogeneous than heart or liver mitochondria in relation to onset of a permeability transition, and that this heterogeneity depends on their Pi transport capacity. Taken together, these results indicate that the inhibitory effects of acidic pH on the PTP may be overcome in situ by an increased rate of Pi uptake, and that ischemic and postischemic acidosis may worsen rather than relieve PTP-dependent tissue damage.