Cells degrade excess and effete organelles by the process of autophagy. Autophagic stimulation of rat hepatocytes by serum deprivation and glucagon (1 µM) caused a fivefold increase of spontaneously depolarizing mitochondria to about 1.5% of total mitochondria after 90 min. Cyclosporin A (CsA, 5 µM), an immunosuppressant that blocks the mitochondrial permeability transition (MPT), prevented this depolarization. Depolarized mitochondria moved into acidic vacuoles labeled by LysoTracker Red. These autophagosomes also increased several‐fold after autophagic stimulation. CsA blocked autophagosomal proliferation, whereas tacrolimus, an immunosuppressant that does not block the MPT, did not. In conclusion, the MPT initiates mitochondrial depolarization after autophagic stimulation and the subsequent sequestration of mitochondria into autophagosomes.