The permeability of the inner mitochondrial membrane from rat liver to D-arginine was studied. By using safranin as a probe of the membrane potential, depolarization of energized liver mitochondria occurred in a dose-dependent fashion starting at 3.3 mmol/L of D- or DL-arginine. When ethidium bromide fluorescence was employed, a decrease in the membrane potential due to D- or DL-arginine was observed. A parallel significant change in succinate-induced respiration in rat liver mitochondria was found in response to osmotic swelling in 125 mmol/L of D-arginine salts. L-Arginine, L-glutamine, L-asparagine, L-ornithine, D-ornithine, and L-lysine did not modify the membrane potential at the concentrations tested. D-Arginine was not transformed into citrulline, but 1.0 mmol/L of the D-amino acid inhibited, by 42%, the state 3 of mitochondrial respiration using succinate as substrate. When D-arginine was used in combination with nigericin, a 40% inhibition of mitochondrial respiration in state 3 was recorded with succinate and with glutamate-malate as substrates.