Background: Pituitary autoantibodies are found in autoimmune hypophysitis and other conditions. They are a marker of pituitary autoimmunity but currently have limited clinical value. The methods used for their detection lack adequate sensitivity and specificity, mainly because the pathogenic pituitary autoantigen(s) are not known and therefore antigen-based immunoassays have not been developed. Objectives: This study aimed to identify novel pituitary autoantigens using sera as probes in proteomic assays. We also compared immunoblotting and immunofluorescence methods for their accuracy in diagnosing autoimmune hypophysitis. Study design and subjects: Twenty-eight sera from autoimmune hypophysitis cases (14 histologically proven and 14 clinically suspected) were compared to 98 sera from controls, which included 14 patients with pituitary adenomas, 48 with autoimmune thyroiditis (15 Graves' disease and 33 Hashimoto's thyroiditis) and 36 healthy subjects. Methods: All sera were tested against human pituitary cytosolic proteins separated by one-dimensional (1D) gel electrophoresis. The band recognition was analysed statistically to detect molecular weight regions preferentially recognized by hypophysitis sera. 2D gel immunoblotting and mass spectrometry were then used to sequence the protein spots of interest. Sera were also tested by immunofluorescence for their recognition of Macaca mulatta pituitary sections. Results: A single region in the 25-27-kDa range was recognized more often by hypophysitis cases than healthy subjects (P = 0.004) or patients with pituitary adenomas (P = 0.044). This region contained two novel candidate autoantigens: chromosome 14 open reading frame 166 (C14orf166) and chorionic somatomammotrophin. Immunoblotting positivity for the 25-27-kDa region yielded greater sensitivity (64% vs. 57%) and specificity (86% vs. 76%) than immunofluorescence in predicting histologically proven hypophysitis, although the performance was still inadequate to make immunoblotting a clinically useful test. Conclusion: The study reports two novel proteins that could act as autoantigens in autoimmune hypophysitis. Further studies are needed to validate their pathogenic role and diagnostic utility.