Lymphocytic hypophysitis is part of the spectrum of organ-specific autoimmune diseases, and although its histopathology is well documented, its pathogenesis is unclear. Serum autoantibodies directed against a 49-kDa cytosolic protein are detected by immunoblotting in 70% of patients with biopsy-proven lymphocytic hypophysitis. Here we report the purification and identification of this first target autoantigen in lymphocytic hypophysitis. The autoantigen has a molecular mass of 49 kDa, a cytosolic localization, and a ubiquitous tissue distribution. The 49-kDa protein was purified from monkey brain and human placental cytosol. Limited amino acid sequencing after proteolytic digestion of the human placental protein showed identity with α-enolase. The identification was confirmed using sera from patients with pituitary autoimmunity, which strongly reacted with recombinant human α-enolase and yeast enolase, but not with rabbit muscle β- enolase. This indicates that the immunoreactive epitopes are largely conserved from yeast to human, but are not present in β-enolase. α-Enolase autoantibodies are not specific to pituitary autoimmune disease and have been reported in other autoimmune diseases. However, this study is the first to indicate a role for α-enolase as an autoantigen in lymphocytic hypophysitis.