Acromegaly is a rare disorder due to an excessive production of growth hormone (GH), typically caused by a GH-secreting pituitary adenoma. Anti-pituitary antibodies (APAs) are often seen in patients with different kinds of pituitary pathologies. Because GH has been proposed as a possible antigen recognized by such antibodies, the prevalence of APAs may be higher in conditions characterized by excessive GH secretion. The primary aim of this study was to compare the prevalence of APAs in patients with acromegaly and in controls with other types of pituitary tumors and healthy subjects. Secondary aim was to characterize the pituitary cells targeted by the APAs. Thirty eight acromegaly patients and 215 controls, including 38 patients with prolactinomas, 64 with non-functioning pituitary adenomas (NFPA), and 113 healthy subjects were enrolled in the study. All subjects were tested for APAs using indirect immunofluorescence. Target cells recognized by APAs were identified by double staining immunofluorescence. APAs were significantly more prevalent in acromegaly cases than in healthy controls (10.5% vs. 1.8%, P < 0.05). This prevalence was similar to that found in patients with prolactinomas (7.9%) and NFPA (12.5%). Among APAs-positive subjects, antibodies recognizing somatotrope cells were more common in acromegaly cases than in healthy controls (3/4 vs. 0/113, P < 0.0001), but had similar frequencies in NFPA (2/8) and prolactinomas (1/3). APAs are more frequently found in patients with pituitary adenomas than healthy subjects, with no significant difference among the tumor types studied. GH-secreting cells could represent a target of the autoimmune response.