Mutations in HERG not only reduce IKr to cause QT syndrome (LQTS) but have also been associated with atrial fibrillation (AF). The mechanisms in AF are unknown. To identify genetic defects conferring AF susceptibility, we screened HERG in 375 patients with typical and lone AF, and identified three probands with rare, non-synonymous HERG variants absent in control populations (284). The first was a C-terminal HERG variant (R1047L), previously reported in LQTS, in 2 probands. One proband was part of a kindred that included 2 other family members with AF or palpitations, and all 3 were mutation carriers; no family was available in the 2nd proband. A second variant (R954C) located only six residues from a previously identified LQTS variant (S960N) was also identified in a lone AF proband. In mutation carriers, QT intervals during sinus rhythm were normal. These variants are particularly interesting because AF and LQTS mutations are likely to be located in close structural proximity. We compared the functional effects of these mutations and WT in two heterologous cell systems: HEK cells stably expressing endogenous HERG (HERG-HEK) or ‘empty’ HEK cells. R1047L caused a 1.4 fold increase in current amplitude in HERG-HEK. In empty HEK cells, there was no difference between R1047L and WT. R954C generated currents that were similar to WT in both HERG-HEK and empty HEK cells, although the nearby S960N variant reduced current 1.6 fold in HERG-HEK and 2 fold in empty HEK cells. These results suggest that relative expression levels of normal and mutant alleles determine net effect on ionic current and action potential controls. Variability in these mechanisms, across or within chambers, may contribute to phenotypes that manifest in only one chamber.