Recent evidence indicates that 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), a marker enzyme of myelin and oligodendrocytes is also present in neural and non-neural mitochondria. However, its role in mitochondria is still completely unclear. We found CNP in rat brain mitochondria (RBM), and studied the effects of CNP substrates, 2',3'-cyclic nucleotides, on functional parameters of RBM. 2',3'-cAMP and 2',3'-cNADP stimulated Ca²⁺ overload-induced Ca²⁺ release from mitochondrial matrix. This Ca²⁺ release under threshold Ca²⁺ load correlated with membrane potential dissipation and mitochondrial swelling. The effects of 2',3'-cyclic nucleotides were suppressed by cyclosporin A, a potent inhibitor of permeability transition (PT). PT development is a key stage in initiation of apoptotic mitochondria-induced cell death. 2',3'-cAMP effects were observed on the RBM functions only when PT was developed. This demonstrates involvement of 2',3'-cAMP in PT regulation in RBM. We also discovered that under PT development the specific enzymatic activity of CNP was reduced. Thus, we hypothesize that suppression of CNP activity under threshold Ca²⁺ load leads to elevation of 2',3'-cAMP levels that in turn promote PT development in RBM. Similar effects of 2',3'-cyclic nucleotides were observed in rat liver mitochondria. Involvement of CNP in PT regulation was confirmed in experiments using mitochondria from CNP-knock-down oligodendrocytes (OLN93 cells). CNP reduction in these mitochondria correlated with lowering the threshold for Ca²⁺ overload-induced Ca²⁺ release. Thus, our results reveal a new function for CNP and 2',3'-cAMP in mitochondria, being a regulator/promotor of mitochondrial PT.