Thiocyanate has been shown to inhibit the following functions in liver mitochondria: Succinate and β-hydroxybutyrate oxidation, P/O ratio, ATP binding, Pi-ATP exchange, and valinomycin-induced K+ uptake. This inhibition can be overcome by increased ADP or substrate concentrations, and is presumably due to interaction of SCN- with anion-binding sites in the mitochondrial membrane. It has been shown (1) that the action of SCN- on the gastric mucosa is not limited to inhibition of HC1 secretion, but appears to extend to effects on K+ permeability and on metabolism since there is alteration in O2 consumption (2) and inhibition of microsomal and mitochondrial ATPase (3). By measurements of the action of SCN-on: (1) P/O ratio of mitochondrial preparations with a variety of substrates; (2) oxidation rate of various substrates; (3) ion transport of mitochondria; and (4) binding of nucleotides by mitochondria, it was shown that SCN- had significant effects on various aspects of mitochondrial metabolism. These results have been presented in preliminary form (4). Methods. Rat liver mitochondria were prepared by differential centrifugation using standard techniques with three additional washings (5), and the purity of the preparation was routinely checked by phase-contrast microscopy. The medium was 0.25 M sucrose, 0.001 M Tris, pH 7.4, and 0.001 M EDTA. The mitochondria were either used fresh, aged for 24 hr at 4°, sonicated for 30 sec at 0°, or treated with digitonin (6). For measurement of oxidative phosphorylation, either the Chance-Williams technique (7), or the glucose-hexokinase method were used. The medium contained 50 mM Tris HCl, pH 7.4, 5 mM MgNO3, 2.5 mM K2HPO4, 0.5 mM EDTA, 2.5 mg/ml albumin, and 55 mM KCl. Rate of oxidation of various substrates at 10 mM was measured using either the Clark electrode or the Warburg apparatus.