This study was performed to determine whether in dogs with pituitary-dependent hyperadrenocorticism (PDH) excessive release of adrenocorticotrophic hormone (ACTH) is accompanied by secretion of ACTH precursor molecules. In addition, we investigated whether the plasma ACTH precursor concentrations were correlated with the size of the pituitary gland and with the degree of resistance to negative glucocorticoid feedback. In 72 dogs with PDH, the plasma ACTH precursor concentration was determined by calculating the difference between the results of a radioimmunoassay (RIA) in which besides ACTH, ACTH precursors were also measured and a highly specific immunoradiometric assay (IRMA) using a polyclonal antibody against ACTH. The degree of resistance to glucocorticoid feedback was established by determining the effect of dexamethasone administration (0.1mg/kg) on the urinary corticoid/creatinine ratio. The pituitary height/brain area (P/B) ratio, determined by computed tomography, was used as a measure for the size of the pituitary gland. The plasma ACTH precursors concentration ranged from 18 to 2233ng/L (median 93ng/L). In 38 dogs, the pituitary was enlarged and plasma ACTH precursors concentrations in these dogs (median 130ng/L, range 24-2233ng/L) were significantly (P<0.05) higher than those in the dogs without pituitary enlargement (median 72ng/L, range 18-481ng/L). In concordance, P/B ratios correlated significantly with plasma ACTH precursor concentrations (r=0.35, P<0.01). In addition, the P/B ratios were significantly correlated with the degree of dexamethasone resistance (r=0.42, P<0.001). Plasma ACTH precursor concentrations in the dexamethasone-resistant dogs (median 210ng/L, range 24-628ng/L) were significantly higher (P<0.01) than those in the dexamethasone-sensitive dogs (median 72ng/L, range 18-2233ng/L). Similarly, the degree of dexamethasone resistance was also significantly correlated with the plasma ACTH precursor concentrations (r=0.33, P<0.01). Dogs with an elevated plasma alpha-MSH concentration (n=14) had significantly (P<0.001) higher plasma ACTH precursor concentrations (median 271ng/L, range 86-2233ng/L) than dogs with non-elevated alpha-MSH (median 73ng/L, range 18-481ng/L). In addition, the plasma concentrations of alpha-MSH correlated significantly with both plasma ACTH precursor concentrations (r=0.53, P<0.001) and P/B ratios (r=0.26, P<0.05). In conclusion, in all dogs with PDH the ACTH concentrations determined by the RIA were higher than the concentrations measured by IRMA indicating the presence of circulating ACTH precursors. High plasma ACTH precursor concentrations were especially found in dexamethasone-resistant dogs with large corticotroph adenomas, some of them probably of PI origin. In the association of large corticotroph adenoma, dexamethasone resistance and high plasma concentrations of ACTH precursors, the decreased sensitivity of the corticotroph cells to glucocorticoid feedback may play a pivotal role.