Cyclooxygenase-2 (COX-2) catalyzes formation of primarily prostaglandin E2 (PGE2). Previous data suggest that intrarenal PGE2 counteracts epithelial Na and water reabsorption and lowers urine concentrating ability. Recent data show that hypothalamic PGE2-EP1 receptors may promote urine concentrating ability through stimulation of vasopressin. We hypothesized that COX-2 promotes urine concentrating ability through stimulation of vasopressin in the hypothalamus. To address this we employed COX-2 deficient (-/- ) and wildtype (WT) littermate mice to metabolic and expression studies of vasopressin-regulated transport proteins. Hypothalamus was examined for vasopressin mRNA and peptide levels as well as PGE2. Basal urine output and water intake was enhanced and urine osmolality was lower in COX-2-/- mice. Upon water deprivation (WD), COX-2-/- displayed a higher plasma osmolality and sodium. Medullary interstitial osmolality and sodium was higher in COX-2-/- in response to WD. Urinary PGE2 excretion was unchanged, but COX-1 protein level was increased in COX-2-/-. Vasopressin mRNA and peptide levels in hypothalamus were increased in COX-2-/- mice compared to WT following WD, however plasma vasopressin was unchanged. Moreover, PGE2 production in brain tissue was increased in COX-2-/- mice. Baseline abundance and apical targeting of vasopressin-regulated transport protein aquaporin-2 (AQP2) was increased in COX-2-/- kidneys. Levels of cAMP, a major regulator of AQP2 trafficking, was unchanged. AQP3 protein was upregulated in COX-2- /- mice and further increased in response to water deprivation. The protein abundance of vasopressin V2 receptor and Na+-K+-2Cl- cotransporter (NKCC2) decreased in COX-2-/- kidney medulla. The data are compatible with the notion that COX- 2 products promote urine concentrating ability through a local renal effect on collecting duct water permeability with compensatory stimulation of hypothalamic vasopressin stores in COX-2-/-.