Background: Lymphocytic hypophysitis is one of the causes of hypopituitarism, which is considered an autoimmune reaction in the anterior pituitary. Method: We examined antipituitary antibodies in patients with lymphocytic hypophysitis and related diseases by immunoblotting method. Results: Autoantibodies to a 22-kDa human pituitary cytosolic protein were identified in significantly higher frequencies in sera from patients with lymphocytic hypophysitis (11 of 15, 73.3%) and isolated ACTH deficiency (7 of 9, 77.8%) compared with Hashimoto thyroiditis, Basedow’s disease and normal control subjects. Also, reactivity against a 49-kDa human pituitary cytosolic protein was seen in 6 of 15 patients (40%) with lymphocytic hypophysitis. N-terminal amino acid sequences of 22-kDa human and rat pituitary cytosolic protein were FPTIPLSVL and FPAMPLSSLFAN, respectively, suggesting that they are human and rat growth hormone, respectively. The pituitary dysfunction (at least one hormone dysfunction) was observed in 11 of 14 patients. Nine of them (82%) showed 22 kDa antibody but 2 of them (18%) did not. Conclusion: The present study demonstrated that pituitary autoantibodies could be involved in the pathogenesis of lymphocytic hypophysitis and could be a positive marker for the disease.