Mitochondrial integrity and function constitute a prerequisite for cellular function and repair processes. We have previously shown that mitochondria of different cell types exhibit pronounced fragmentation under hypothermic conditions. This fission, accompanied by a decline of cellular ATP content, showed reversibility at 37^◦C. However, it is unclear whether other temperatures as currently discussed for reconditioning of organs allow this reconstitution of mitochondria. Therefore, we here study in a model of cultured porcine aortic endothelial cells how different rewarming temperatures affect mitochondrial re-fusion and function. After 48 h cold incubation of endothelial cells in Krebs-Henseleit buffer with glucose (5 mM) and deferoxamine (1 mM) at 4^◦C pronounced mitochondrial fission was observed. Following 2 h rewarming in cell culture medium, marked fission was still present after rewarming at 10^◦ or 15^◦C. At 21^◦C some re-fusion was visible, which became more marked at 25^◦C. Networks of tubular mitochondria similar to control cells only re-appeared at 37^◦C. ATP content decreased at 4^◦C from 3.6 ± 0.4 to 1.6 ± 0.4 nmol/10⁶ cells and decreased even further when rewarming cells to 10^◦ and 15^◦C. Values after rewarming at 21^◦C were similar to the values before rewarming while ATP gradually increased at higher rewarming temperatures. Metabolic activity dropped to 5 ± 11% of control values during 4^◦C incubation and recovered with increasing temperatures to 36 ± 10% at 25^◦C and 78 ± 17% at 37^◦C. Integrity of monolayers, largely disturbed at 4^◦C (large gaps between endothelial cells; cell injury ≤ 1%), showed partial recovery from 15^◦C upwards, complete recovery at 37^◦C. Endothelial repair processes (scratch assay) at 25^◦C were clearly inferior to those at 37^◦C. These data suggest that reconditioning temperatures below 21^◦C are not optimal with regard to reconstitution of mitochondrial integrity and function. For this goal, temperatures of at least 25^◦C appear required, with 30^◦C being superior and 37^◦C yielding the best results.