The interaction of diazoxide, a diabetogenic compound inhibiting tricarboxylic acid cycle oxidations, with the energy conservating system of mitochondria was studied. Changes of respiratory activity of livermitochondria with various substrates and corresponding changes of the redox states of respiratory pigments after addition of diazoxide were measured. The flux of reducing equivalents into the respiratory chain and the potential of the “energized state” is drastically lowered by the inhibitor. The drug developed only a weak uncoupling activity but a pronounced inhibition on substrate uptake. The interaction of diazoxide with accumulation of 14C-labelled substrate anions was directly measured, and is of the competitive type. Diazoxide itself enters the mitochondrion in the anionic form, as revealed by anion exchange studies. ATP-ase is activated by diazoxide. This effect is reversed by sulfonylureas and is discussed as a mode for the antagonism of both compounds at the cellular membrane of pancreatic islets.