The effects, of the benzodiazepines RO5-4864, AHN 086, PK 11195 and clonazepam on respiration of mitochondria from heart, kidney, and liver were studied. ADP-stimulated respiration of heart mitochondria was the most sensitive to inhibition by AHN 086; clonazepam was not inhibitory. Several respiratory chain segment activities of submitochondrial particles were insensitive to AHN 086, except for NADH oxidase which was partially inhibited. However, in contrast to submitochondrial particles, the succinate-cytochrome c oxidoreductase activity in intact mitochondria was inhibited by AHN 086, suggesting an effect at the substrate transport level. Phosphate-induced, succinate-dependent swelling was also inhibited by AHN 086 it was not affected by clonazepam. Uncoupled ATP hydrolysis was partially inhibited by RO5-4864, AHN 086, and clonazepam. It is suggested that there is an unspecific inhibition of NADH oxidase and ATP hydrolysis by these benzodiazepines and a specific inhibition on oxidizable substrate transport by the peripheral-type benzodiazepine AHN 086.