Previous reports that ethacrynic acid and furosemide inhibit the respiration of isolated mitochondria suggested a direct action of these diuretics on oxidative metabolism. To explore this possibility further, the effects of ethacrynic acid and furosemide on the oxygen consumption of mitochondria isolated from the cortex and outer medulla of rat kidneys were investigated. Both diuretics inhibited state 3 and uncoupled respiration supported by glutamate-malate (which enters the electron transport chain prior to site I) and succinate (which enters prior to site II); respiration supported by tetramethyl phenylene diamine-ascorbate (which enters prior to site III) was relatively unaffected. Biochemical bypass of site II significantly alleviated the respiratory inhibition by both agents. Confirmation of these findings was provided by measurement of the electron transport carriers by dual wavelength spectroscopy, which showed that both diuretics caused a reduction of flavoproteins and an oxidation of the cytochromes. It is concluded that ethacrynic acid and furosemide inhibit oxidative phosphorylation in vitro by inhibiting electron transport through phosphorylation site II.