The UT-A1 and UT-A3 urea transporters are expressed in the terminal inner medullary collecting duct (IMCD) and play an important role in the production of concentrated urine. We have shown that both hyperosmolarity and vasopressin increase urea permeability in perfused rat terminal IMCDs, and that UT-A1 and UT-A3 accumulate in the plasma membrane in response to vasopressin. In this study we investigated whether hyperosmolarity causes UT-A1 and/or UT-A3 to accumulate in the plasma membrane or represents a complimentary stimulatory pathway. Rat IMCD suspensions were incubated in 450 vs. 900 mOsM solutions. We biotinylated the IMCD surface proteins, collected and analyzed them. Membrane accumulation was assessed by western blotting of the biotinylated protein pool probed with anti-UT-A1 or anti-UT-A3. We studied the effect of NaCl, urea, and sucrose as osmotic agents. Membrane associated UT-A1 and UT-A3 increased relative to control levels when either NaCl (UT-A1 increased 37±6%; UT-A3 increased 46±13%) or sucrose (UT-A1 increased 81±13%; UT-A3 increased 60±8%) were used to increase osmolarity. There was no increase in membrane UT-A1 or UT-A3 when urea was added. Analogously, UT-A1 phosphorylation was increased in NaCl- and sucrose- but not in urea- based hyperosmolar solutions. Hypertonicity also increased UT-A3 phosphorylation. We conclude that the increase in the urea permeability in response to hyperosmolarity reflects both UT-A1 and UT-A3 movement to the plasma membrane and may be a direct response to tonicity. Furthermore, this movement is accompanied by, and may require, increased phosphorylation in response to hypertonicity.