Isolated adrenocorticotropin deficiency (IAD) is characterized by low or absent adrenocorticotropic hormone (ACTH) production. IAD is presumed to be caused in part by an autoimmune mechanism, and several lines of evidence have suggested the presence of anti-pituitary antibodies in IAD. However, the exact autoantigens remain unknown. The present study was designed to identify the autoantigen(s) in IAD using chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Rat anterior pituitary lysate was subjected to SDS-PAGE, and immunoblotting was performed using the sera from two patients with IAD and from a healthy subject. The bands detected by the patient serum samples, but not by the healthy subject sample, were excised, in-gel digested using trypsin, and subjected to LC-MS/MS analysis. On immunoblots, a 51-kDa band in the insoluble pellet was detected by the sera from the IAD patients but not from the healthy subject. Mass spectrometric analysis revealed the 51-kDa band contained Rab guanine nucleotide dissociation inhibitor (GDI) alpha. Consistent with the mass spectrometric analysis, a recombinant full-length human Rab GDI alpha was recognized by the two IAD patient samples but not by the healthy subject sample using immunoblotting. In total, anti-Rab GDI alpha antibodies were detected in serum samples from three of five patients with IAD (60%) but were absent in 5 healthy subjects. In addition, Rab GDI alpha was expressed in the anterior pituitary. In conclusion, it appears that Rab GDI alpha is a candidate autoantigen involved in IAD, and that anti-Rab GDI alpha antibodies are present predominantly in patients with IAD.