When isolated rat liver mitochondria are incubated in KCI medium, matrix volume, flux, and forces in both hypo- and hyperosmolarity are time-dependent. In hypoosmotic KCl medium, matrix volume is regulated via the K+/H+ exchanger. In hyperosmotic medium, the volume is regulated in such a manner that at steady state, which is reached within 4 min, it is maintained whatever the hyperosmolarity. This regulation is Pi- and deltamuH+-dependent, indicating Pi-K salt entry into the matrix. Under steady state, hyperosmolarity has no effect on isolated rat liver mitochondria energetic parameters such as respiratory rate, proton electrochemical potential difference, and oxidative phosphorylation yield. Hypoosmolarity decreases the NADH/NAD+ ratio, state 3 respiratory rate, and deltamuH+, while oxidative phosphorylation yield is not significantly modified. This indicates kinetic control upstream the respiratory chain. This study points out the key role of potassium on the regulation of matrix volume, flux, and forces. Indeed, while matrix volume is regulated in NaCl hyperosmotic medium, flux and force restoration in hyperosmotic medium occurs only in the presence of external potassium.