Endothelial cells (EC) exhibit a rise in cytosolic calcium (Cai) and a formation of intercellular gaps during ischemia. Both parameters aggravate during reoxygenation (Reox) and this is accompanied by endothelial barrier failure. As a consequence, edema appears in the affected organ, e.g. the heart, leading to malfunction. The aim of this study was to characterise the impact of mitochondria in mediating this Cai increase. Microvascular coronary EC from humanely killed rats were exposed to simulated ischemia (40min, pH 6.4) followed by Reox (40min, pH 7.4, 2.5mM glucose). Cai was monitored via fura 2, ATP loss was determined via the Mag-Fura-2 method, mitochondrial membrane potential (mMP) was analysed via JC-1 and gaps were identified planimetrically. The Fo/F1-ATPase inhibitor oligomycin (OGM, 10µM) and the mitochondria-specific scavenger Mito Q (1µM) were applied during Reox. The data presented describe the mean values ± s.e.m. in arbitrary units of fluorescent intensity. They were taken from at least 4 different experiments. Under control conditions, the fura 2 ratio increased after 40 min of Reox significantly to 1.43±0.01 (p<0.05 vs. end-anoxia 1.25±0.02, n=140 cells). Gap formation increased to 240% ±20% (p<0.05 vs. end-anoxia 100%±1%; n=140 cells). During ischemia the JC-1 ratio decreased significantly from 1.02±0.08 to an end-anoxic value of 0.60±0.06 (p<0.05, n=80 cells) indicating a mMP depolarisation. Within the first 5 min of Reox the JC-1 ratio went back to normoxic level, but decreased again to 0.77±0.06 after 40 min of Reox (p<0.05 vs. normox., n=80 cells). During ischemia the Mag-Fura-2 ratio increased from 1.00 ± 0.00 to an end-anoxic value of 1.12 ± 0.01 pointing to an ATP loss (p<0.05 vs. normox., n=184 cells). With the onset of Reox the Mag-Fura-2 ratio decreased initially to almost basal level indicating an ATP recovery and then rose continuously to 1.16 ± 0.01 (n=184 cells) again pointing to an increasing ATP loss. Application of OGM, acting as an inhibitor of the Fo/F1-ATPase, reduced Cai after 40 min of Reox (1.36 ± 0.02 vs. w/o OGM, p<0.05; n=140 cells) and also reduced the ATP loss significantly (1.11 ± 0.01 vs. 1.16 ± 0.01 w/o OGM, p<0.05). The formation of gaps was also blocked by OMG (112% ±8% vs. 240% ±20% p<0.05). Application of Mito Q blocked the Cai rise (1.19 ± 0.08 vs. w/o Mito Q, p<0.05; n=120 cells) and reduced the ATP loss significantly (1.08 ± 0.02 w/o Mito Q, p<0.05; n=100 cells). In conclusion: During Reox Cai rises and a formation of gaps occur while the mitochondria depolarise and ATP is reduced. Increasing the cellular ATP content by inhibiting the Fo/F1-ATPase reduces both Cai rise and formation of gaps. The loss of ATP is possibly mediated by production of mitochondrial ROS since Mito Q improves both, ATP loss and Cai rise.