Cyclosporin A (CsA) is a widely used compound because of its potent immunosupressive properties, derived mainly from the inhibition of calcineurin, and also because of its ability to block the mitochondrial permeability transition pore (PTP). This second effect has been involved in the protection against apoptosis mediated by release of mitochondrial factors. We show here that CsA (1–10μM) has an additional effect on Ca2+ homeostasis in mitochondria that cannot be attributed to inhibition of PTP. By measuring specifically mitochondrial [Ca2+] with targeted aequorin, we show that CsA inhibited Ca2+ entry into mitochondria both in intact and in permeabilized cells, and this effect was stronger when Ca2+ entry was triggered by low cytosolic [Ca2+], below 5 μM. Inhibition of mitochondrial Ca2+ uptake required micromolar concentrations of CsA and was not mimicked by other inhibitors of calcineurin such as FK-506 or cypermethrin, nor by a different inhibitor of the PTP, bongkrekic acid. CsA blocked the increase in mitochondrial Ca2+ uptake rate induced by the mitochondrial Ca2+ uniporter activator SB202190. Our results suggest that CsA inhibits Ca2+ entry through the Ca2+ uniporter by a mechanism independent of the inhibition of PTP or calcineurin. This effect may contribute to reduce depolarization and Ca2+ overloading in mitochondria after cell stimulation, and thus cooperate with the direct inhibition of PTP to prevent apoptosis.Financial support from Dirección General de Enseñanza Superior, Ministerio de Ciencia y Tecnología (BFI2002-01397) and from Junta de Castilla y León (VA 005/02) are gratefully acknowledged.Peer reviewed