The structure of isolated rat liver mitochondria has been observed in the electron microscope following incubation of the mitochondria in vitro under a variety of conditions. The results show that ultrastructural changes are only associated with the energization and deenergization of isolated mitochondria if the composition of the incubation medium permits ion movements in or out of the matrix. The mechanism of energy coupling does not appear to depend on these major mitochondrial structural changes. The addition of low levels of valinomycin greatly increases the rate at which the matrix compartment swells and shrinks on energization and deenergization even at low K+ concentrations.