AVP increases the osmotic water permeability of renal collecting ducts by inducing the translocation of specific aquaporin-2 (AQP2) water channels from cytoplasmic vesicles to the apical plasma membrane of the principal cells. Here, we report a novel inner medullary tubule suspension for the study of this phenomenon that overcomes some of the drawbacks faced by present techniques; both primary cultures of inner medullary collecting duct cells and cell lines expressing AQP2 show aberrant trafficking and/or signaling pathways. The tubule suspensions were prepared by proteolytic digestion of inner medullas dissected from freshly isolated rat kidneys. After drug treatment, cellular distribution of AQP2 was determined by membrane fractionation and Western blotting or by immunocytochemistry. Treatment of suspensions with 1 nM AVP caused redistribution of AQP2 to the apical plasma membrane of the principal cells, a process inhibited by microtubule disruption or PKA inhibition. We conclude that this method provides a valuable new approach to the study of the cellular mechanisms involved in the response of the collecting duct to AVP.