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Pituitary adenomas are commonly reported in older horses. The typical clinical signs associated with this condition, also known as equine Cushing's disease (ECD), are related to increased adrenocorticotropin (ACTH) production resulting in hyperadrenocorticism. The primary purpose of this study was to determine whether plasma ACTH concentrations differed between cushingoid and healthy horses. The second objective was to determine the effects of blood sample handling techniques on ACTH concentrations. A commercial human ACTH radioimmunoassay (RIA) was used to quantify equine plasma ACTH. Intra-assay and interassay variations, as well as dilutional parallelism were determined during the RIA validation. Plasma ACTH concentrations were evaluated in a group of healthy equids composed of 18 horses and 9 ponies, and in 22 equids with a clinical diagnosis of hyper adrenocorticism (11 horses and 11 ponies). The mean plasma ACTH concentrations in healthy horses and ponies, (18.68 ± 6.79 pg/mL (mean ± SD) and 8.35 ± 2.92 pg/mL, respectively), were significantly different (P = .009). The mean plasma ACTH concentration in horses and ponies with ECD, (199.18 ± 182.82 pglmL and 206.21 ± 319.56 pg/mL, respectively), were significantly higher than the mean ACTH concentration in the control animals (P < .001). Plasma ACTH concentrations appeared to be a sensitive and specific indicator of ECD in horses and ponies. ACTH concentrations measured in plasma samples kept at room temperature (19°C) as long as 3 hours after blood collection were not statistically different from those of samples kept at 1°C. J Vet Intern Med 1996;10:1–6. Copyright 1996 by the American College of Veterinary Internal Medicine.