Abstract When rat liver mitochondria were incubated for 15 min at 37° and pH 7.4 in 250 mm sucrose, 50 mm tris(hydroxymethyl)aminomethane hydrochloride, 10 mm MgCl2, 10 mm inorganic orthophosphate, and 1 mm ethylenediaminetetraacetate, the resulting loss of endogenous K+ was greatly increased by the addition of 2.5 to 10 mm ammonium sulfate or sodium sulfate to the incubation medium. The increased rate of K+ loss, which could be completely prevented by the addition of ATP, succinate, or glutamate to the medium, began 4 to 8 min after the start of incubation. Ammonium sulfate and sodium sulfate also increased the loss of K+ when the mitochondria were first suspended at 0° in a solution containing KCl before being added to the incubation medium. When KCl was added to the mitochondria after 15 min of incubation at 37°, both the passive uptake and the active uptake of K+ were not affected by the simultaneous addition of either 10 mm ammonium sulfate or sodium sulfate. However, when mitochondria were incubated for 15 min with ammonium sulfate before the addition of KCl, active uptake of K+ appeared to be abolished. In the absence of exogenous Pi, sodium sulfate inhibited the loss of endogenous K+, while ammonium sulfate still caused an increased K+ loss. In the absence of exogenous Mg++, both ammonium sulfate and sodium sulfate inhibited the loss of endogenous K+ whether or not Pi was present in the medium. Addition of sulfate ions to the incubation medium also increased the leakage from mitochondria of compounds showing an absorption maximum at 259 mµ.