Studies were performed to examine urinary diluting ability and protein abundance of renal aquaporins and ion transporters in glucocorticoid deficient (GD) rats at baseline and in response to oral water loading. All rats underwent bilateral adrenalectomies followed by aldosterone replacement (GD) or combined aldosterone and dexamethasone replacement (CTL). Prior to oral water loading, GD rats demonstrated significantly decreased urine output and increased urinary osmolality as compared to CTL. These findings were accompanied by a significant upregulation of the protein abundance of inner medulla AQP2 (148 ± 18%), phosphorylated AQP2 (p-AQP2) (156 ± 13%), and AQP3 (145 ± 8%) in GD rats as compared with CTL (all p values were less than 0.05). GD rats also demonstrated a marked reduction in urinary sodium excretion compared to pair-fed CTL. There was a significant upregulation of NKCC2, NHE3 and cortical -, -ENaC subunits in the GD rats. One hour following an acute water load of 40 ml/kg by oral gavage, GD rats demonstrated decreased percent water excretion (5 ± 1% vs. 33 ± 9%, p<0.01), decreased urine output (33 ± 12 vs. 250 ± 65 µl/kg/min, p<0.05) and higher Uosm (1894 ± 292 vs. 316 ± 92 mOsmol/kg H2O, p<0.001) than CTL. These findings were accompanied by increased plasma vasopressin level (1.6 ± 0.2 vs. 0.9 ± 0.2 pg/ml, p<0.05), protein expression of inner medullary AQP2 (149 ± 5%), p-AQP2 (177 ± 9%) (p values were less than 0.01) and maintained apical expression of AQP2 in GD as compared to CTL rats. Treatment with the vasopressin V₂ receptor antagonist, OPC-31260, increased percent water excretion and urine output and reduced U_osm when compared with vehicle-treated GD rats. The increased abundance and apical trafficking of inner medulla AQP2 and p-AQP2 protein in GD rats was also reversed with the V₂ receptor antagonist. In conclusion, enhanced protein abundance of sodium transporters and sodium channels with sodium retention occurred with glucocorticoid deficiency. Most strikingly, V₂ receptor antagonist administration resulted in reversal of upregulation and apical trafficking of AQP2 and p-AQP2 in association with improved urinary diluting capacity and increased water excretion following oral water loading.