The neuronal protein p42IP4 was suggested to be involved in neurodegenerative processes. To find out a role of p42IP4 in cell death, we detected apoptosis in control and p42IP4 overexpressing mouse neuroblastoma (N2a) cells by caspase-3 assay, DNA-laddering assay and flow cytometry analysis. We have shown that p42IP4 is not involved in apoptosis. However, we observed that p42IP4 had an effect on cell cycle. Cellular Ca2+ signals are crucial in the control of most physiological processes, cell injury and cell death. Mitochondria play a central role in cellular Ca2+ signalling. During cellular Ca2+ overload, mitochondria take up cytosolic Ca2+, which, in turn, can lead to opening of the permeability transition pore (PTP). Although Ca2+-dependent PTP has been implicated in a broad range of cell death pathways, the exact mechanism of the PTP opening remains elusive. Previously, p42IP4 was identified in brain membrane fraction, which also contained mitochondria. Some other data indicate possible localization of p42IP4 in mitochondria. Therefore, this important question was studied here. We determined for the first time mitochondrial localization of p42IP4. Moreover, in rat brain mitochondria (RBM), we found interaction of p42IP4 with 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) and [alpha]-tubulin by pull-down binding assay and by immunoprecipitation. Localization of p42IP4 and CNP in the inner membrane fraction of mitochondria prompted us to study whether p42IP4 and CNP are involved in regulation of mitochondrial Ca2+-induced PTP. Simultaneous measurements of the respiratory rate, trans-membrane potential, and Ca2+ transport in the mitochondrial suspension were performed. We determined the rate of Ca2+ influx, Ca2+ capacity and lag time for PTP opening in mitochondria isolated from p42IP4-transfected and from control N2a cells. Overexpression of p42IP4 led to promotion of Ca2+-induced PTP opening. Furthermore, p42IP4 ligands, phosphatidylinositol(3,4,5)trisphosphate and inositol(1,3,4,5)tetrakisphosphate, accelerated PTP opening in mitochondria isolated from N2a cells. We found the interaction of CNP with modulators of PTP, adenine nucleotide transporter and voltage-dependent anion channel. The enzymatic activity of CNP was reduced under PTP opening. Involvement of CNP in PTP operation was confirmed in experiments using mitochondria isolated from CNP-knock-down oligodendrocyte cell line (OLN93). In mitochondria isolated from OLN93 cells transfected with CNP-targeting small interfering RNA, CNP reduction was correlated with facilitation of Ca2+-induced PTP opening. The CNP substrates, 2’,3’- cyclic AMP and 2’,3’- cyclic NADP, enhanced PTP development in RBM. In summary, our results suggest that p42IP4 and CNP in mitochondria play a role in regulation of mitochondrial Ca2+ transport mechanisms in the brain. While Ca2+-induced PTP opening is important stage of initiation of cell death, consequently we hypothesize that in the brain p42IP4 and CNP contribute to processes leading to neurodegenerative diseases.