Aging is accompanied by progressive dysfunction of mitochondria associated with a continuous decrease of their capacity to produce ATP. Mitochondria isolated from brain of aged animals show an increased mitochondrial permeability transition pore (mPTP) opening. We recently detected new regulators of mPTP function in brain mitochondria, the enzyme 2′, 3′–cyclic nucleotide 3′–phosphodiesterase (CNP) and its substrates 2′, 3′–cAMP and 2′, 3′–cNADP, and the neuronal protein p42IP4. Here, we compared parameters of mPTP opening in non-synaptic brain mitochondria isolated from young and old rats. In mitochondria from old rats (>18 months), mPTP opening occurred at a lower threshold of Ca2+ concentration than in mitochondria from young rats (<3 months). mPTP opening in mitochondria from old rats was accelerated by 2′, 3′–cAMP, which further lowered the threshold Ca2+ concentration. In non-synaptic mitochondria from old rats, the CNP level was decreased by 34%. Lowering of the CNP level in non-synaptic mitochondria with aging was accompanied by decreased levels of voltage-dependent anion channel (VDAC; by 69%) and of p42IP4 (by 59%). Thus, reduced levels of CNP in mitochondria could lead to a rise in the concentration of the mPTP promoter 2′, 3′–cAMP. The level of CNP and p42IP4 and, probably VDAC, might be essential for myelination and electrical activity of axons. We propose that in aging the reduction in the level of these proteins leads to mitochondrial dysfunction, in particular, to a decreased threshold Ca2+ concentration to induce mPTP opening. This might represent initial steps of age-related mitochondrial dysfunction, resulting in myelin and axonal pathology.