Vasopressin-induced exocytosis was dissected in native and aquaporin-2-expressing (AQP2) renal LLC-PK₁ cells using a fluorimetric exocytosis assay based on soluble secreted yellow fluorescent protein (ssYFP). YFP was targeted to the secretory pathway by adding an 18 amino acid signal peptide from hen egg-white lysozyme. Immunofluorescence labeling together with analysis of Alexa-555-dextran internalization revealed that ssYFP is exclusively located in the secretory pathway. Both immunofluorescence and immunogold electron microscopy showed significant co-localization between ssYFP and AQP2. Fluorimetry and western blot analysis demonstrated similar constitutive ssYFP secretion in native LLC-PK₁ cells and cells expressing AQP2. In AQP2-expressing cells, a two-fold increase in ssYFP secretion was observed within 15 min of VP stimulation. This transient burst of ssYFP secretion was abolished by the PKA inhibitor H-89, and was not seen in native cells. The endocytotic inhibitor methyl-ß-cyclodextrin, which also promotes membrane accumulation of AQP2, had no effect on ssYFP secretion. While cells expressing phosphorylation-deficient AQP2-S256A showed significantly lower baseline levels of constitutive secretion, VP still induced a significant increase in exocytosis. Our data indicate that: a) this assay can monitor exocytosis in cultured epithelial cells; b) VP has an acute stimulatory effect on ssYFP secretion in cells expressing AQP2 but not in native cells; c) phosphorylation of AQP2 at S256 may be involved in the regulation of constitutive AQP2 exocytosis, and play only a minor role in the VP-induced burst. These results support the idea that VP increases AQP2 exocytosis in addition to its role in reducing AQP2 endocytosis.