In order to investigate the mechanism of action of ethacrynic acid and furosemide, experiments were designed to determine whether these drugs directly inhibit active transport or energy metabolism. The effects of these diuretics on the respiration of tubule suspensions isolated from renal cortex (of rats and rabbits) and outer medulla (of rabbits) were measured. The respiration of tubules prepared from renal outer medulla was stimulated by the presence of chloride in the incubation medium, whereas the respiration of cortical tubules was unaffected by chloride. Both ethacrynic acid and furosemide produced the greatest inhibition of respiration on tubules from outer medulla suspended in chloride-containing media; this result suggests that the diuretics directly inhibit chloride transport. The source of metabolic energy for ion transport was varied by using substrates which donate electrons to the respiratory chain at different phosphorylation sites. Both ethacrynic acid and furosemide inhibit respiration supported by beta-hydroxybutyrate, but there was little or no inhibition of respiration with succinate or tetramethylphenylenediamine ascorbate. Similarly, ouabain inhibited respiration with beta-hydroxybutyrate, but not with the other substrates. Therefore, both diuretics inhibited respiration in a fashion similar to ouabain. It is concluded from both types of experiments that ethacrynic acid and furosemide may directly inhibit active chloride transport.