Cytosolic Ca²⁺ concentration is maintained at a level approximately 10 000-fold lower than that in the extracellular media, and is strictly regulated within defined limits by the interplay of Ca²⁺ channels, pumps and exchangers. The mitochondria and endoplasmic reticulum (ER) play a significant role in this process by storing and buffering Ca²⁺. Ca²⁺ also regulates mitochondrial respiration, and is involved in protein synthesis and folding within the ER. Certain pathophysiological conditions, including apoptosis and neuronal excitotoxicity, have been ascribed to a sustained high intracellular Ca²⁺ concentration. In this study, we investigated whether the structure of intracellular organelles in HeLa cells is affected by prolonged increases in cytosolic Ca²⁺. Application of a Ca²⁺ ionophore (ionomycin, 10 µM) to intact cells bathed in buffer containing 1.8 mM calcium resulted in the breakdown in the structure of mitochondria and the ER in 100 % of cells (n = 110 from 3 experiments). Confocal imaging of cells expressing ER- or mitochondrially targeted red/green fluorescent proteins (DsRed2 and EGFP) revealed that mitochondria initially swell, fragment and then undergo permeability transition as determined by calcein release in response to elevated Ca²⁺. Simultaneous with the fragmentation of mitochondria, the ER undergoes a dramatic vesicularisation followed by contraction towards the nucleus. In contrast, the Golgi and nuclear envelope were unaffected. The Ca²⁺-induced breakdown of mitochondrial and ER membranes occurred in a concentration-dependent manner (EC₅₀ ~30 µM) (10 cells per coverslip, 3 coverslips per calcium concentration from 1 to 1000 µM). In addition, the response was time dependent, in that a lower (~1 µM) Ca²⁺ concentration could cause organelle disruption if applied for periods greater that 30 min (n = 20 cells from 3 experiments). The effect was due to Ca²⁺ elevation rather than a non-specific consequence of ionomycin application, since the ionophore had no effect when extracellular Ca²⁺ was absent. Furthermore, the fragmentation of intracellular organelles could also be achieved by stimulation of heterologously expressed NMDA receptors (100 µM glutamate). The microtubule network was also fragmented by Ca²⁺ elevation. However, stabilisation of the microtubules with Taxol (10 µM for 30 min) failed to prevent organelle fragmentation. The fragmentation, however, was inhibited by application of Trolox (100 µM for 1 h), a lipid peroxidation inhibitor, or of MnTBaP (200 µM, 30 min), a superoxide dismutatase mimetic (3 coverslips from 3 experiments). This indicated that organelle fragmentation may occur as a result of lipid peroxidation occurring downstream of calcium elevation. Inhibition of calpains and caspases did not prevent the calcium-induced fragmentation. The disruption of organelles during elevated Ca²⁺ signals may contribute to the death of cells under pathological conditions, such as excessive NMDA receptor activity during neuronal excitotoxicity.