We have previously demonstrated that activating autoantibodies to β1-adrenergic receptor (β1AR) and M2 muscarinic receptor (M2R) facilitate atrial fibrillation (AF) in patients with Graves' disease (GD). The objectives of this expanded study were to examine the prevalence of β1AR, β2AR, and M2R autoantibodies in hyperthyroidism subjects. Sera from 81 patients including 31 with GD and AF, 36 with GD and sinus rhythm, 9 with toxic multinodular goiter, 5 with subacute thyroiditis, and 10 control subjects were examined for these autoantibodies by ELISA. Sera from 20 ELISA-positive GD subjects, 10 with AF and 10 with sinus rhythm, were assayed for autoantibody bioactivity using cell-based bioassays. In patients with GD and AF, 45, 65, and 77 % were ELISA positive for β1AR, M2R, and β2AR autoantibodies, respectively. In patients with GD and sinus rhythm, 17, 39, and 75 % were ELISA positive for β1AR, M2R, and β2AR autoantibodies, respectively. β1AR and M2R autoantibodies were co-present in 39 % of patients with GD and AF compared to 14 % in GD with sinus rhythm (p = 0.026). Patients with toxic multinodular goiter or subacute thyroiditis had a low prevalence of autoantibodies. The mean β1AR and M2R autoantibody activity was elevated in both GD groups but higher in those with AF than those with sinus rhythm. β2AR autoantibody activity was also increased in both groups. In conclusion, β1AR, β2AR, and M2R autoantibodies were elevated in GD. β1AR and M2R autoantibodies appear to be related to concurrent AF, while β2AR autoantibodies were equally prevalent in those with a sinus tachycardia and those with AF.