The mixed ion channel blocker, vernakalant (RSD1235), is effective in rapid conversion of atrial fibrillation (AF) to sinus rhythm (SR). Suppression of cardiac two-pore-domain potassium (K2P) channels causes action potential prolongation and has recently been proposed as a novel antiarrhythmic strategy. The objective of this study was to investigate acute effects of vernakalant on human K2P2.1 (TREK-1) and K2P3.1 (TASK-1) channels to provide a more complete picture of its antiarrhythmic mechanism of action. The class IC antiarrhythmic drug flecainide was studied as a comparator agent. Two-electrode voltage clamp and whole-cell patch clamp electrophysiology was used to record K2P currents from Xenopus oocytes and Chinese hamster ovary (CHO) cells. Vernakalant inhibited cardiac K2P2.1 channels expressed in Xenopus oocytes and in CHO cells. The IC50 value obtained from mammalian cells (13.3 µM) was close to the range of vernakalant levels reported in patients (2-8 µM), indicating potential clinical significance of K2P2.1 blockade. Open rectification characteristics and current-voltage relationships of K2P2.1 currents were not affected by vernakalant. Vernakalant did not significantly reduce K2P3.1 currents. Finally, the class I antiarrhythmic drug flecainide had no effect on K2P2.1 or K2P3.1 channels. In conclusion, the recently developed antiarrhythmic drug vernakalant targets human K2P2.1 K(+) background channels. This previously unrecognized inhibitory property adds to the multichannel blocking profile of vernakalant and extends the mechanistic basis for its anti-fibrillatory effect.