Adrenocortical steroid hormones are importantly involved in the regulation of extracellular fluid volume. The present study was aimed at examining whether aldosterone and/or glucocorticoid regulates the abundance of aquaporin-3 (AQP3), -2, and -1 in rat kidney. In protocol 1, rats were adrenalectomized, followed by aldosterone replacement, dexamethasone replacement, or combined aldosterone and dexamethasone replacement (rats had free access to water but received a fixed amount of food).Protocol 2 was identical to protocol 1, except that all groups received fixed daily food and water intake. In bothprotocols 1 and 2, aldosterone deficiency was associated with increased fractional Na excretion and severe hyperkalemia. Semiquantitative immunoblotting revealed that aldosterone deficiency was associated with a dramatic downregulation of AQP3 abundance. Consistent with this, immunocytochemistry and immunoelectron microscopy revealed a marked decrease in AQP3 labeling in the basolateral plasma membranes of collecting duct principal cells. In contrast, AQP1 and AQP2 abundance and distribution were unchanged. Glucocorticoid deficiency revealed no changes in AQP3, -2, or -1 abundance. In protocol 3, Na restriction (to increase endogenous aldosterone levels) or exogenous aldosterone infusion in either normal rats or vasopressin-deficient Brattleboro rats was associated with a major increase in AQP3 abundance. In protocol 4, aldosterone levels were clamped by infusion of aldosterone, while Na intake was altered from a low to a high level. Under these circumstances, there were no changes in AQP3 or AQP2 abundance, although the level of the thiazide-sensitive Na-Cl cotransporter was decreased. In conclusion, the results uniformly demonstrate that aldosterone regulates AQP3 abundance independently of Na intake. In contrast, changes in glucocorticoid levels in these models do not influence AQP3 or AQP2 abundance. Therefore, in the collecting duct aldosterone may regulate, at least in part, AQP3 expression in addition to regulating Na and K transport.