In a forward genetic screen, a mouse with an Aqp2 mutation was identified. The purpose of this study was to characterize this murine model of recessive nephrogenic DI. We now report a novel F204V mutation in the Aqp2 gene. This allele of Aqp2 was found to cause the first mouse model of NDI to survive past the first week of life. Molecular analyses concluded that mutant AQP2 adopts a different subcellular localization in renal collecting-duct cells, and was resistant to translocation induced by desmopressin, an agonist of AVP. In vitro studies using the Madin-Darby canine kidney (MDCK) cell line demonstrated an endoplasmic reticulum pattern for the mutant protein, and apparent resistance to translocation. These data conclusively prove that autosomal recessive NDI is a consequence of improper AQP2 routing in the intact mammal.