Generally most intracellular Ca²⁺is stored in the endoplasmic reticulum (ER) and mitochondria. Recently a mitochondrial Ca²⁺-induced Ca²⁺release (mCICR) mechanism, unconnected with ryanodine receptors (RyR's), has been shown in tumour cells. The existence of a mitochondrial Ca²⁺release mechanism in BAE cells was investigated using saponin-permeabilised BAE cells. When buffered intracellular solutions were ‘stepped’ from 10 nM to 10 μM free Ca²⁺, the mitochondrial inhibitors CN (2 mM), FCCP (1 μM), and RR (20 μM) significantly reduced total CICR by approximately 25%. The ER Ca²⁺-ATPase inhibitor thapsigargin (100 nM) had no effect. Furthermore, cyclosporin A (200 nM), an inhibitor of the mitochondrial permeability transition pore (PTP), abolished total CICR. Therefore, the novel ryanodine-caffeine insensitive CICR mechanism previously reported in BAE cells involves mitochondrial Ca²⁺release. It is proposed that in BAE cells, mCICR occurs via the mitochondrial PTP and may be physiologically important in endothelial cell Ca²⁺signalling.