The canonical transient receptor potential channels, TRPC3 and TRPC6, are abundantly expressed along with the water channel aquaporin-2 (AQP2) in principal cells of the cortical and medullary collecting duct. Although TRPC3 is selectively localized to the apical membrane and TRPC6 is found in both the apical and basolateral domains, immunofluorescence is often observed in the cytoplasm suggesting that TRPC3 and TRPC6 may exist in intracellular vesicles and may shuttle to and from the membrane in response to receptor stimulation. To test this hypothesis, the effect of arginine-vasopressin (AVP) on the subcellular distribution of TRPC3, TRPC6 and AQP2 was examined in rat kidney, and in cultured cell lines from the cortical (M1) and inner medullary (IMCD-3) collecting duct. Immunofluorescence analysis revealed that TRPC3, but not TRPC6, co-localized with AQP2 in intracellular vesicles. AVP caused the insertion and accumulation of TRPC3 and AQP2 in the apical membrane, but had no effect on the subcellular distribution of TRPC6. TRPC3, but not TRPC6, co-immunoprecipitated with AQP2 from both medulla and M1 and IMCD-3 cell lysates. Apical-to-basolateral transepithelial ⁴⁵Ca²⁺ flux in polarized IMCD-3 cell monolayers was stimulated by diacylglycerol analogs or by the purinergic receptor agonist, ATP, but not by thapsigargin. Stimulated ⁴⁵Ca²⁺ flux was increased by over-expression of TRPC3 and attenuated by a dominant-negative TRPC3 construct. Furthermore, ⁴⁵Ca²⁺ flux was greatly reduced by the pyrazole-derivative BTP2, a known inhibitor of TRPC3 channels. These results demonstrate that 1) TRPC3 and TRPC6 exist in different vesicle populations, 2) TRPC3 physically associates with APQ2 and shuttles to the apical membrane in response to AVP, and 3) TRPC3 is responsible for transepithelial Ca²⁺ flux, in principal cells of the renal collecting duct. Key Words: Ion channels, renal nephron, immunoprecipitation, immunofluorescence, subcellular localization, Ca²⁺ flux.