Hyperosmotic stress can be encountered by the kidney, the skin and during treatment of acute brain damage. It can lead to cell cycle arrest or apoptosis. Exactly how mammalian cells detect hyperosmolarity and how the cell chooses between cell cycle arrest or death remains to be established. It has been proposed that hyperosmolarity is detected directly by growth factor receptor protein-tyrosine kinases. To investigate this we asked whether growth factors and osmotic stress cooperate in the activation of signaling pathways. Receptors responded normally to the presence of growth factors and we observed normal levels of GTP-bound Ras under hyperosmotic conditions. In contrast, activation of Raf, Akt, Erk-1, Erk-2 and Jun N-terminal kinase were strongly reduced. These observations suggest that hyperosmotic conditions block signaling directly downstream of active Ras. It is thought that apoptotic cell death due to environmental stress is initiated by cytochrome c release from the mitochondria. Visualization of cytochrome c by immunofluorescence showed that hypertonic conditions result in a break-up of the mitochondrial network, which is re-established within one hour after replacing hypertonic with isotonic medium. When we carried out live imaging, we observed that the mitochondrial membrane potential disappears immediately after the onset of hyperosmotic shock. Our observations provide new insights into the hypertonic stress response pathway. In addition, they show that signaling down stream of Ras and mitochondrial dynamics can easily be manipulated by the exposure of cells to hyperosmotic conditions.