K⁺ channels in the basolateral membrane of mouse cortical collecting duct (CCD) principal cells were identified with patch-clamp technique, real-time PCR, and immunohistochemistry. In cell-attached membrane patches, three K⁺ channels with conductances of ∼75, 40, and 20 pS were observed, but the K⁺ channel with the intermediate conductance (40 pS) predominated. In inside-out membrane patches exposed to an Mg²⁺-free medium, the current-voltage relationship of the intermediate-conductance channel was linear with a conductance of 38 pS. Addition of 1.3 mM internal Mg²⁺ had no influence on the inward conductance (G_in = 35 pS) but reduced outward conductance (G_out) to 13 pS, yielding a G_in/G_out of 3.2. The polycation spermine (6 × 10⁻⁷ M) reduced its activity on inside-out membrane patches by 50% at a clamp potential of 60 mV. Channel activity was also dependent on intracellular pH (pH_i): a sigmoid relationship between pH_i and channel normalized current (NP_o) was observed with a pK of 7.24 and a Hill coefficient of 1.7. By real-time PCR on CCD extracts, inwardly rectifying K⁺ (Kir)4.1 and Kir5.1, but not Kir4.2, mRNAs were detected. Kir4.1 and Kir5.1 proteins cellularly colocalized with aquaporin 2 (AQP2), a specific marker of CCD principal cells, while AQP2-negative cells (i.e., intercalated cells) showed no staining. Dietary K⁺ had no influence on the properties of the intermediate-conductance channel, but a Na⁺-depleted diet increased its open probability by ∼25%. We conclude that the Kir4.1/Kir5.1 channel is a major component of the K⁺ conductance in the basolateral membrane of mouse CCD principal cells.