The current study aimed to investigate the midterm (24 hour) response of 17-hydroxyprogesterone (17-OHP) and dehydroepiandrosterone sulphate (DHEA-S) to synthetic high-dose adrenocorticotropin (ACTH) in adrenal incidentalomas (AI). Seventeen patients with AI and 40 age- and sex-matched controls received synthetic ACTH (tetracosactide, 1000 μg, IM). Plasma, 17-OHP and DHEA-S were collected in basal conditions and after 1, 4, 6, 8 and 24 hours. (HPA) axis was also evaluated using circadian serum cortisol, urinary free cortisol and over-night 2 mg dexamethasone suppression. Basal plasma 17-OHP levels did not differ among the groups. However, the increment in plasma 17-OHP in patients both in terms of peak [13.76±2.52, 4.77±0.30 ng/ml, mean±S.E.M, p<0.001] and area under the curve [190±46, 96.75±32 ng/ml/h, p<0.001] were significantly higher than that of the controls. Stimulated 17OH-P levels never reached 9.1 ng/ml in controls. Sixty-five (11/17) % of the patients were found to have exaggerated response. Three of the patients were found to have subclinical Cushing's syndrome and interestingly, two augmented their 17-OHP response to ACTH after unilateral adrenalectomy and normalisation of their HPA axis. Basal DHEA-S levels of the patients were significantly lower [99.21±45, 230.18±34 μg/dl, p<0.01] and stayed persistently lower than that of the controls. Evidence of a heterozygous 21 hydroxylase deficiency, as indicated by the exaggerated 17-OHP response to ACTH, has been widely reported in AI patients. However, to our knowledge to date there is no report on augmented 17-OHP response to ACTH after adrenalectomy. Possible reasons for the augmentation were discussed.