Aims: Atrial fibrillation (AF) is the most common cardiac arrhythmia. Disturbances in cardiac potassium conductance are considered as one of the disease mechanisms in AF. We aimed to investigate if mutations in potassium-channel β-subunits KCNE2 and KCNE3 are associated with early-onset lone AF. Methods & results: The coding regions of KCNE2 and KCNE3 were bidirectionally sequenced in 192 unrelated patients diagnosed with early-onset lone AF (<40 years). Two nonsynonymous missense mutations were identified in KCNE2 (M23L and I57T). Both mutations were absent in a healthy control group (n = 1500 alleles). Electrophysiological investigations were performed for both mutations in combination with candidate pore-forming α-subunits KV7.1, KV11.1, KV4.3 and KV1.5. A significant gain-of-function effect was observed upon coexpression with KV7.1 and KV7.1 + KCNE1. Confocal imaging found no differences in subcellular localization. No disease-suspected mutations were identified in KCNE3. Conclusion: We identified two KCNE2 gain-of-function missense mutations that seem to increase the susceptibility of early-onset lone AF. These results confirm previous findings indicating that gain-of-function in the slow delayed rectifier potassium current might be involved in the pathogenesis of AF.