Triggering of the permeability transition pore (PTP) in isolated mitochondria causes release of matrix Ca²⁺, ions, and metabolites, and it has been proposed that the PTP mediates mitochondrial Ca²⁺ release in intact cells. To study the role of the PTP in mitochondrial energy metabolism, the mitochondrial content of Ca²⁺, Mg²⁺, ATP, and ADP was determined in hormonally stimulated rat livers perfused with cyclosporin A (CsA). Stimulation of livers perfused in the absence of CsA with glucagon and phenylephrine induced an extensive uptake of Ca²⁺, Mg²⁺, and ATP plus ADP by the mitochondria, followed by a release on omission of hormones. In the presence of CsA, the PTP was fully inhibited, but neither the hormone-induced uptake of Ca²⁺, ATP, or ADP by mitochondria nor their release after washout of hormones was significantly changed. We conclude that the regulation of sustained changes in mitochondrial Ca²⁺ content induced by hormonal stimulation is independent of the PTP.