Large conductance Ca²⁺-activated K⁺ channels (BK), comprised of pore-forming and accessorysubunits, secrete K⁺ in the distal nephron under high flow and high K⁺ diet conditions. BK channels are detected by electrophysiology in many nephron segments; however, the accessory subunit associated with these channels has not been determined. We performed RT-PCR, Western blotting and immunohistochemical staining to determine whether BK-1 is localized to the connecting tubule's principal-like cells (CNTs) or intercalated cells (ICs), and whether BK-2-4 are present in other distal nephron segments. Reverse transcription (RT)-PCR and Western blots revealed that the mouse kidney expresses BK-1, BK-2 and BK-4. Available antibodies in conjunction with BK-1^-/- and BK-4^-/- mice allowed the specific localization of BK-1 and BK-4 in distal nephron segments. Immunohistochemical staining showed that BK- 1 is localized in CNTs, but not ICs of the connecting tubule. The localization of BK-4 was discerned using an anti-BK-4 antibody on wild type tissue and anti-GFP on GFP-replaced BK-4 (BK-4^-/-) mouse tissue. Both antibodies (anti-BK-4 and anti-GFP) localized BK-4 to the thick ascending limb (TAL), distal convoluted tubule (DCT), and ICs of the distal nephron. It is concluded that BK-1 is narrowly confined to the apical membrane of CNTs in the mouse, whereas BK-4 is expressed in TAL, DCT and ICs.