In brain, p42IP4 (centaurin-α1; recently named ADAP 1, which signifies ADP ribosylation factor GTPase activating protein with dual PH domains 1, within the large family of Arf-GTPase activating proteins) is mainly expressed in neurons. p42IP4 operates as a dual receptor recognising two second messengers, the soluble inositol(1,3,4,5)tetrakisphosphate and the lipid phosphatidylinositol(3,4,5)trisphosphate. We show here for the first time that p42IP4 is localized in mitochondria, isolated from rat brain and from cells transfected with p42IP4. In rat brain mitochondria we additionally found interaction of p42IP4 with 2′, 3′-cyclic nucleotide 3′-phosphodiesterase and α-tubulin by pull-down binding assay and by immunoprecipitation. In mitochondria from Chinese hamster ovary cells, p42IP4 is predominantly associated with the intermembrane space and the inner membrane. This localization of p42IP4 indicates that p42IP4 might have a still unknown mitochondrial function. We studied whether p42IP4 is involved in Ca2+-induced permeability transition pore opening, which is important in mitochondrial events leading to programmed cell death. We used mouse neuroblastoma cells as a model for the functional studies of p42IP4 in mitochondria. In mitochondria isolated from p42IP4-transfected mouse neuroblastoma cells, over-expression of p42IP4 significantly decreased Ca2+ capacity and lag time for Ca2+ retention. Thus, we suggest that p42IP4 is involved in the regulation of Ca2+ transport in mitochondria. We propose that p42IP4 promotes Ca2+-induced permeability transition pore opening and thus destabilizes mitochondria.