Atrial natriuretic peptide (ANP) is known to acutely promote water and sodium excretion whereas subchronic effects involve water retention. Renal hemodynamics and water and sodium excretion were determined in response to continuous ANP infusion in conscious rats where body sodium and fluid balance was constantly maintained by servo-controlled replacements. Moreover the subcellular localization of aquaporin-2 (AQP2) and epithelial sodium channel (ENaC) subunits was determined at 10 or 90 min after ANP infusion. ANP (0.5 µg/kg/min) evoked a transient (peak at 10 min) but significant diuresis (5 fold) followed by a reduced urine production to control levels. Consistent with this the fractional distal water excretion was significantly increased in the first 30 min and then decreased in a later phase of 30-90 min after infusion of ANP. Immunocytochemistry revealed absence of changes in the subcellular localization of AQP2 and p-AQP2 (AQP2 phosphorylated in the PKA consensus site S256) at 10 min after ANP infusion. In contrast, at 90 min of ANP infusion a marked increase in apical labeling of AQP2 and p-AQP2 was observed in inner medullary collecting duct principal cells and a detectable increase in outer medullary collecting duct cells, but not in cortical collecting ducts. This was further supported by in vitro studies showing that ANP induced plasma membrane insertion of AQP2 in transiently AQP2-transfected HEK 293 cells. ANP evoked an instant increase in renal sodium excretion and the increased urinary Na excretion and fractional distal Na excretion persisted during 90 min after ANP infusion albeit a slightly lower level compared to that observed during the first 30 min. At 10 min there were no observable changes in the subcellular localization of ENaC subunits. In contrast, markedly increased apical targeting of - and -subunits was observed at 90 min of infusion but not of the -ENaC subunit. In conclusion 1) ANP infusion induced a sustained natriuresis and transient diuresis. 2) There were no changes in the subcellular localization of AQP2 and ENaC subunits in the early phase (10 min of infusion). 3) In contrast, ANP infusion induced a marked increase in apical targeting of AQP2, p-AQP2, - and -ENaC 90 min after ANP infusion. The increased apical targeting of ENaC subunits and AQP2 is likely to represent direct or compensatory effects to increase sodium and water reabsorption to prevent the development of volume depletion in response to ANP infusion.