The intrarenal autocrine-paracrine dopamine (DA) system mediates a significant fraction of the natriuresis in response to a salt load. DA inhibits a number of Na+ transporters to effect sodium excretion, including the proximal tubule Na+/H+ exchanger-3 (NHE3). DA represent a single hormone that regulates NHE3 at multiple levels including translation, degradation, endocytosis, and protein phosphorylation. Since cell surface NHE3 protein is determined by the balance between exocytotic insertion and endocytotic retrieval, we examined whether DA acutely affects the rate of NHE3 exocytosis in a cell culture model. DA inhibited NHE3 exocytosis at a dose-dependent manner with a half maximal around 10-6 M. The DA effect on NHE3 exocytosis was blocked by inhibition of protein kinase A (PKA) and by Brefeldin A, which inhibits endoplasmic reticulum-to-Golgi transport. NHE3 directly interacts with the coatomer protein COP based on yeast-two-hybrid and co-immunoprecipitation. Since NHE3 has been shown to be recycled back to the cell membrane after endocytosis, we measured NHE3 recycling using a biochemical re-insertion assay and showed that re-insertion of NHE3 back to the membrane is also inhibited by DA. In conclusion, amongst the many mechanisms by which DA reduce apical membrane NHE3 and induces proximal tubule natriuresis, one addition mechanism is inhibition of exocytotic insertion and re-insertion of NHE3 into the apical cell surface.