It is recognized that dopamine promotes natriuresis by inhibiting multiple transporting systems in the proximal tubule. In contrast, less is known about molecular targets of dopamine actions on the water-electrolyte transport in the cortical collecting duct (CCD). Epithelial cells in the CCD are exposed to dopamine which is synthesized locally or secreted from sympathetic nerve endings. Basolateral K⁺ channels in the distal renal tubule are critical for potassium recycling and controlling basolateral membrane potential to establish driving force for Na+ reabsorption. We demonstrate that K_ir4.1 and K_ir5.1 are highly expressed in the mouse kidney cortex and are localized to the basolateral membrane of the CCDs. Using patch clamp electrophysiology in freshly isolated CCDs, we detected highly abundant 40 pS and scarce 20 pS single channel conductances most likely representing K_ir4.1/5.1 and K_ir4.1 channels, respectively. Dopamine reversibly decreased open probability of both channels having a relatively greater action on K_ir4.1/5.1 heterodimer. This effect was mediated by D2-like but not D1-like dopamine receptors. Protein kinase C blockade abolished inhibition of the basolateral K⁺ channels by dopamine. Importantly, dopamine significantly decreased the amplitude of K_ir4.1/5.1 and K_ir4.1 unitary currents. Consistently, dopamine induced an acute depolarization of basolateral membrane potential as was directly monitored using current clamp mode in isolated CCD. Therefore, we demonstrate that dopamine inhibits basolateral K_ir4.1/5.1 and K_ir4.1 channels in CCD cells via stimulation of D2-like receptors and subsequently PKC. This leads to depolarization of basolateral membrane and a decreased driving force for sodium reabsorption in the distal renal tubule.