Serum anti-pituitary antibodies (APAs) to cytosolic antigens have been found in association with autoimmune hypophysitis, idiopathic hypopituitarism, and other autoimmune endocrinopathies. Here, an immunoblot method was used to search for serum autoantibody (AAb) reactivities against pituitary antigens, including nuclear and cytoskeletal proteins, in six patients with idiopathic hypopituitarism, 60 patients with type 1 diabetes, nine patients with autoimmune polyglandular syndrome (APS) type 1, and in 74 healthy controls. Frequent patient serum IgG reactivity was observed against a 60 kDa human pituitary antigen, and the cross-reactive 62 kDa protein from rat brain was identified as alpha-internexin (alpha-INX) by proteomic methods. IgG and IgM AAbs to this neuron-specific type IV intermediate filament (IF) protein were found in most sera of patients with endocrine autoimmunity as well as healthy subjects with no significant differences in frequencies between the groups, but the levels of IgM alpha-INX AAbs were higher in patients with hypopituitarism as compared to healthy controls (P = 0.032, Mann-Whitney U-test). These findings suggest that alpha-INX AAbs are not specifically related to autoimmune endocrine diseases and most probably are a part of the natural AAb repertoire. This is the first demonstration of alpha-INX AAbs as one of the predominant neuronal IF AAbs in human sera.