Cultured renal epithelial cells rapidly downregulate expression of the vasopressin-regulated water channel aquaporin-2 (AQP-2). Our aim was to define conditions that favor maintenance of AQP-2 expression in vitro without genetic manipulation. We show here that primary cultures of rat inner medullary collecting duct (IMCD) cells retain AQP-2 expression for at least 6 days when grown with dibutyryl cAMP (DBcAMP) supplementation. We also found that coating the culture dishes with type IV collagen, rather than rat-tail collagen, retards AQP-2 downregulation. Immunofluorescence and biochemical studies indicate a shuttling of AQP-2-bearing vesicles after stimulation with vasopressin or forskolin. Rab3 proteins, known to be involved in regulated exocytosis, were detected only in cells grown in the presence of DBcAMP. Using the adenylyl cyclase assay, we confirmed the functional integrity of the vasopressin V2 receptor in a broken cell preparation. Our data show that cAMP supplementation is sufficient for the maintenance of AQP-2 expression in primary cultured cells. The model system established here allows the study of the regulation of genes encoding the antidiuretic machinery at the cellular level.