AVP and atrial natriuretic peptide (ANP) have opposite effects in the kidney. AVP induces antidiuresis by insertion of aquaporin-2 (AQP2) water channels into the plasma membrane of collecting duct principal cells. ANP acts as a diuretic factor. An ANP- and nitric oxide (NO)/soluble guanylate cyclase (sGC) induced insertion of AQP2 into the plasma membrane is reported from different models. However, functional data on the insertion of AQP2 is missing. We used primary cultured inner medullary collecting duct (IMCD) cells and digital holographic microscopy, calcein quenching measurements and immunofluorescence and western blotting to analyze the effects of ANP and NO donors on AQP2 phosphorylation, membrane expression and water permeability. While AVP led to acceleration in osmotically induced swelling, ANP had no effect. However, in AVP pretreated cells ANP decreased significantly the kinetic of cell swelling. This effect was mimicked by 8-bromo-cGMP (8-Br-cGMP) and blunted by protein kinase G (PKG) inhibition. Stimulation of the NO/sGC pathway or direct activation of sGC with BAY 58-2667 had similar effects like ANP. In cells treated with AVP AQP2 was predominantly localized in the plasma membrane, after additional incubation with atrial natriuretic peptide AQP2 was mostly localized in the cytosol indicating an increased retrieval of AQP2 from the plasma membrane by ANP. Western blot analysis showed that ANP was able to reduce AVP-induced phosphorylation of AQP2 at position S256. In conclusion, we show that the diuretic action of ANP or NO in the IMCD involves a decreased localization of AQP2 in the plasma membrane which is mediated by cGMP and PKG.