Lipid rafts are cholesterol- and shingolipid-enriched membrane microdomains implicated in membrane signalling and trafficking. To assess renal epithelial raft functions through the characterization of their associated membrane proteins, we have isolated lipid rafts from rat kidney by sucrose gradient fractionation after detergent treatment. The low density fraction was enriched in cholesterol, sphingolipid, and flotillin-1 known as lipid raft markers. Based on proteomic MALDI-Q-TOF analysis of the low density fraction, the protein with the highest significance score was the alpha subunit of Na⁺,K⁺-ATPase (NKA), whose raft association was validated by simultaneous immunoblotting. NKAb was copurified from the low density fraction. To test the role of lipid rafts in sorting and membrane delivery of renal transporting epithelia, we have chosen to study thick ascending limb (TAL) epithelium for its high NKA activity and the property to be stimulated by antidiuretic hormone (ADH). Cultured rbTAL cells were studied. Cholesterol depletion as well as detergent extraction at warm caused a shift of NKA to the higher density fractions. Comparative preparations from blood monocytes revealed the absence of NKA from rafts in these non-polarized cells. Short term exposure of rbTAL cells to ADH (1h) caused translocation and enhanced raft association of NKA via cAMP activation. Preceding cholesterol depletion prevented this effect. TAL-specific, GPI-anchored Tamm Horsfall protein was copurified with NKA in the same raft fraction suggesting functional interference between these products. These results may have functional implications regarding the turnover, trafficking, and regulated surface expression of NKA as the major basolateral ion transporter of TAL.