The favorable effect of fish oils rich in n-3 polyunsaturated fatty acids (PUFAs) on the development of atrial fibrillation (AF) is controversial. The relationship between the serum concentrations of n-3 PUFAs and the incidence of AF is unclear; therefore, in the present study, we aimed to elucidate this relationship. We evaluated the serum concentrations of n-3 PUFAs in 110 patients with AF, 46 patients with ischemic heart disease (IHD) and no AF, and 36 healthy volunteers. Thirty-six patients had a history of IHD (IHD-AF group) and 74 did not (L-AF group). The eicosapentaenoic acid (EPA) levels in the L-AF group were higher than those in the IHD-AF and control groups (117 ± 64, 76 ± 30, and 68 ± 23 μg/ml, respectively); the docosahexaenoic acid (DHA) levels showed the same pattern (170 ± 50, 127 ± 27, and 126 ± 35 μg/ml, respectively). In both the L-AF and IHD-AF groups, the EPA levels in patients with persistent and permanent AF were higher than those in patients with paroxysmal AF (L-AF 131 ± 74 vs. 105 ± 51 μg/ml; IHD-AF 82 ± 28 vs 70 ± 33 μg/ml). Multivariate analysis showed that cases of AF were associated with higher levels of EPA but not DHA. In this Japanese population study, the EPA and DHA levels in patients with L-AF were higher than those in normal subjects. In particular, the EPA level was associated with the incidence of AF. These findings suggest that an excess of EPA might be a precipitating factor of AF.