Background The acetylcholine activated inward rectifier potassium current I KACh has been suggested to play a role in atrial fibrillation (AF). Therefore, I KACh reduction was proposed as an antifibrillatory intervention. Chloroquine is an antimalarial quinoline shown to be a potent anti-AF agent due, in part, to its I KACh blocking properties. We hypothesize that chloroquine decreases I KACh via 2 different mechanisms: pore block and reduction of Kir3.1/3.4 cell surface expression. Methods and Results Patch-clamp experiments in HEK293 cells stably expressing Kir3.1 and Kir3.4 (GIRK1/4 cells) showed that acute chloroquine application blocked I KACh with an IC 50 of 1.15 μM ± 0.102 (n = 5). In isolated Langendorff perfused mouse hearts, optical mapping showed that 10 μM chloroquine perfusion for 10 minutes increased atrial APD 60 in the presence of 10 μM acetylcholine, from 14.5 ± 1.73 ms to 23 ± 2.16 ms (n = 4, P P P Conclusions Our data suggest that I KACh reduction is achieved via both direct block of the channel and decrease in surface channel expression. Trafficking of Kir3.x and/or its underlying pathways may be an attractive target for anti-AF therapy.