Background - The molecular mechanism of increased background inward rectifier current (I_K1) in atrial fibrillation (AF) is not fully understood. We tested whether constitutively active acetylcholine (ACh)-activated I_K,ACh contributes to enhanced basal conductance in chronic AF (cAF). Methods and Results - Whole-cell and single-channel currents were measured with standard voltage-clamp techniques in atrial myocytes from patients with sinus rhythm (SR) and cAF. The selective I_K,ACh blocker tertiapin was used for inhibition of I_K,ACh. Whole-cell basal current was larger in cAF than in SR, whereas carbachol (CCh)-activated I _K,ACh was lower in cAF than in SR. Tertiapin (0.1 to 100 nmol/L) reduced I_K,ACh in a concentration-dependent manner with greater potency in cAF than in SR (-logIC₅₀: 9.1 versus 8.2; P<0.05). Basal current contained a tertiapin-sensitive component that was larger in cAF than in SR (tertiapin [10 nmol/L]-sensitive current at -100 mV: cAF, -6.7±1.2 pA/pF, n=16/5 [myocytes/patients] versus SR, -1.7±0.5 pA/pF, n=24/8), suggesting contribution of constitutively active I _K,ACh to basal current. In single-channel recordings, constitutively active I_K,ACh was prominent in cAF but not in SR (channel open probability: cAF, 5.4±0.7%, n=19/9 versus SR, 0.1±0.05%, n=16/9; P<0.05). Moreover, I_K1 channel open probability was higher in cAF than in SR (13.4±0.4%, n=19/9 versus 11.4±0.7%, n=16/9; P<0.05) without changes in other channel characteristics. Conclusions - Our results demonstrate that larger basal inward rectifier K⁺ current in cAF consists of increased I_K1 activity and constitutively active I _K,ACh. Blockade of I_K,ACh may represent a new therapeutic target in AF.