An accurate diagnosis of autonomous cortisol secretion (ACS) is important but the specificity of cortisol ≥50 nmol/l after overnight dexamethasone suppression (cortisolONDST ) is low. Therefore, ACTH following overnight dexamethasone suppression (ACTHONDST ) and cortisol following a 2-day dexamethasone suppression test (cortisol2-DAYDST ) were examined as markers of HPA-axis suppression during ONDST.This cross-sectional study examined patients with adrenal incidentalomas and basal ACTH ≥2.0 pmol/l. ACTHONDST /ACTH ratio (ACTH-ratio) was calculated. To define cut-off levels for ACTHONDST and ACTH-ratio as markers of HPA-axis suppression, ROC curves were used to separate patients with cortisolONDST <50 and ≥50 nmol/l.CortisolONDST was ≥50 nmol/l in 140 out of 373 patients. In patients with cortisolONDST <50 nmol/l, ACTHONDST was 0.28 pmol/l (<0.23-2.7). DHEAS was positively correlated to ACTHONDST , demonstrating a 9% increase with a doubling in ACTHONDST , p=0.02. The best cut-off levels for ACTHONDST and ACTH-ratio to detect cortisolONDST ≥50 nmol/ were ≥0.6 pmol/l and ≥18% respectively. These cut-off levels were tested on patients with cortisolONDST <50 nmol/l, considered to have adequate suppression (n=233), and patients with reduction of ≥50 nmol/l from cortisolONDST to cortisol2-DAYDST , who were considered to have inadequate suppression (n=16). ACTHONDST ≥0.6 pmol/l and ACTH-ratio ≥18% had a sensitivity of 75% and 81% respectively, and a specificity of 78% and 85% respectively, for detecting patients with inadequate suppression.ACTHONDST and ACTH-ratio can be markers of HPA-axis suppression in the investigation of adrenal incidentalomas. CortisolONDST ≥50 nmol/l with ACTHONDST <0.6 pmol/l or ACTH-ratio <18% should lead to the suspicion of ACS.