The adenine nucleotide translocase (ANT), besides transferring ATP from the mitochondrial matrix to the rest of the cell, has also been proposed to be involved in mitochondrial permeability transition (MPT), and accordingly in mitochondrial Ca2+ homeostasis. In order to assess the role of ANT in Ca2+ signal transmission from the endoplasmic reticulum (ER) to mitochondria, we overexpressed the various ANT isoforms and measured the matrix [Ca2+] ([Ca2+]m) increases evoked by stimulation with IP3-dependent agonists. ANT overexpression reduced the amplitude of the [Ca2+]m peak following Ca2+ release, an effect that was markedly greater for ANT-1 and ANT-3 isoforms than for ANT-2. Three further observations might explain these findings. First, the effect was partially reversed by treating the cells with cyclosporine A, suggesting the involvement of MPT. Second, the effect was paralleled by alterations of the 3D structure of the mitochondria. Finally, ANT-1 and ANT-3 overexpression also caused a reduction of ER Ca2+ loading that caused a marginal decrease in the cytosolic Ca2+ responses. Overall, these data provide evidence for the involvement of ANT-1 and ANT-3 in the induction of MPT and indicate the relevance of this phenomenon in ER-mitochondria Ca2+ transfer.