Medullary collecting duct function was studied using the in vivo microcatheterization technique in three groups of rats receiving amiloride, hydrochlorothiazide, or both diuretics. In each group of animals, atrial natriuretic factor (ANF99-126) was given in the second phase of the experiment. The combination of amiloride and hydrochlorothiazide resulted in a more marked natriuresis than either diuretic given as a single agent. Sodium reabsorption in the medullary collecting duct, as a fraction of the delivered load, was reduced from 64% (amiloride) and 69% (hydrochlorothiazide) to 29% (amiloride and hydrochlorothiazide). Atrial natriuretic factor reduced collecting duct sodium reabsorption when added to amiloride or hydrochlorothiazide to 23% and to 41%, respectively, but had no additional effect when given with amiloride and hydrochlorothiazide. Potassium excretion with amiloride and hydrochlorothiazide was intermediate between amiloride or hydrochlorothiazide given as single agents. With the diuretic combination, potassium transport showed no significant reabsorption or secretion along the medullary collecting duct, amiloride was associated with potassium reabsorption, and hydrochlorothiazide was associated with potassium secretion in the duct. The results confirm the importance of the medullary collecting duct as a site of diuretic action. The known additive effects of amiloride and hydrochlorothiazide on sodium excretion and the opposing effects of these agents on potassium excretion occur, to a major degree, in the medullary collecting duct. Furthermore, the additive effects of amiloride and ANF indicate that blocking of amiloride-sensitive sodium channels is not the only mechanism of action of ANF on duct salt transport in vivo.