Cytokines, such as interferons (IFN), underlie many immunological functions and are increasingly implicated in disease-related symptoms and pathology. In order to study the potential roles of IFN α and its antagonists in autoimmune phenomena, the sera from 89 patients (aged 15–95 years, 65 females) diagnosed as having myasthenia gravis (MG) (2 months to 34 years duration) were tested for the presence of natural anti-IFN α-2b auto-antibodies. Sera were screened for anti-IFN α-2b by a sandwich-type enzyme immunoassay system. Ten (11.2%) and 6 (6.7%) sera were identified that contained positive-competing and non-competing anti-IFN α-2b auto-antibodies, respectively. The MG sera were further analyzed by immunobloting against reduced IFN α-2b and for neutralizing anti-IFN α activity in an antiviral assay cells system. From tested EIA positive-competing sera, 5 were shown to be positive by immunoblot and 6 sera were found to contain neutralizing anti-IFN α-2b. Four of the 6 neutralizing anti-IFN α-2b sera came from patients with thymoma-associated MG. The sera were studied for linear epitope recognition on the IFN α-2b molecule by a solid phase binding assay, in which overlapping peptides homologous with the entire IFN α-2b sequence were separately synthesized on a nitrocellulose sheet. Peptides number 2 (residues 8–21), 3 (15–28), 6 (33–46), 10 (63–76), 15 (98–112), and 21 (141–154) were immunoreactive. Peptide 21 was apparently associated with antiviral activity, although peptide 21 has not been previously described as an immunogenic determinant on the IFN α-2b molecule. These results indicate that neutralizing anti-IFN α-2b is often present in MG, particularly in cases of thymoma-associated MG, and recognize a variety of epitopes on the IFN α-2b molecule, including those involved in its biological activity. Two groups of IFN epitopes were described associated with patient's age but not with diseases evolution.