Apart from gating by interaction with βγ subunits from heterotrimeric G proteins upon stimulation of appropriate receptors, Kir.3 channels have been shown to be gated by intracellular Na+. However, no information is available on how Na+-dependent gating affects endogenous Kir3.1/Kir3.4 channels in mammalian atrial myocytes. We therefore studied how loading of adult atrial myocytes from rat hearts via the patch pipette filling solution with different concentrations of Na+ ([Na+]pip) affects Kir3 current. Surprisingly, in a range between 0 and 60 mm, Na+ neither had an effect on basal inward-rectifier current nor on the current activated by acetylcholine. Overexpression of Kir3.4 in adult atrial myocytes forced by adenoviral gene transfer results in formation of functional homomeric channels that interact with βγ subunits upon activation of endogenous muscarinic receptors. These channels are activated at [Na+]pip≥ 15 mm, resulting in a receptor-independent basal inward rectifier current (Ibir). Ibir was neither affected by pertussis toxin nor by GDP-β-S, suggesting G-protein-independent activation. PIP2 depletion via endogenous PLC-coupled α1 adrenergic receptors causes inhibition of endogenous Kir3.1/3.4 channel currents by about 75%. In contrast, inhibition of Na+-activated Ibir amounts to < 20%. The effect of the Kir3 channel blocker tertiapin-Q can be described using an IC50 of 12 nm (endogenous IK(ACh)) and 0.61 nm (Ibir). These data clearly identify Ibir as a homotetrameric Kir3.4 channel current with novel properties of regulation and pharmacology. Ibir shares some properties with a basal current recently described in atrial myocytes from an animal model of atrial fibrillation (AF) and AF patients.