Mitochondrial permeability transition is a process marked by a significant increase in the permeability of the inner mitochondrial membrane that can be initiated by massive Ca2+ influx into mitochondria with or without oxidative stress. The increase in membrane permeability is due to induction of a gigantic proteinaceous pore in the inner mitochondrial membrane called the permeability transition pore (PTP). The detrimental role of PTP induction in neuronal mitochondria in various neuropathologies is well documented. There are different methods to study PTP in isolated mitochondria and in live cells. In the present chapter, we describe a method to monitor PTP induction in individual isolated brain mitochondria. Although this method is not often used, it could be an excellent addition to the arsenal of methodologies utilized in studies of PTP. In addition, we present here detailed description of a method used by us to isolate rat or mouse brain nonsynaptic and synaptic mitochondria.