Background: Hyperosmotic stress causes cell death through activation of apoptotic pathways if the protective osmolyte response is impaired. In this study we attempt to elucidate the molecular mechanisms of hypertonicity-induced apoptosis and the effect of major organic osmolytes upon those. Methods: Hypertonicity-induced changes in Bcl2-family protein abundance and the presence of cytochrome c and apoptosis inducing factor (AIF) in the cytoplasm, were measured using western blot and immunofluorescence labeling. To determine dissipation of mitochondrial membrane potential (Δ &Psi) though the permeability transition pore (PTP), the lipophilic cationic carbocyanine fluorescence probe JC-1 and TMRM fluorescence probes were used. Results: Hypertonic culture conditions increase the abundance of proapoptotic Bax and the concentration of cytochrome c and apoptosis inducing factor (AIF) in the cytoplasm. These changes are associated with a dissipation of Δ &Psi and increased permeability of the PTP. We further show that organic osmolytes stabilize the Δ &Psi and decrease the concentration of cytochrome c and AIF in the cytoplasm. Conclusion: Our study shows that organic osmolytes prevent hypertonicity-induced apoptosis by preventing dissipation of Δ &Psi through stabilization of the PTP. These findings further support the important role of organic osmolytes in preventing hypertonicity-mediated cell death in medullary kidney cells.