BACKGROUND DEMONSTRATION OF CENTRAL: Peripheral adrenocorticotropic hormone (ACTH) gradient is important for diagnosis of Cushing's disease. AIM THE AIM WAS TO ASSESS THE UTILITY OF INTERNAL JUGULAR VEIN (IJV): Peripheral vein ACTH ratio for diagnosis of Cushing's disease. MATERIALS AND METHODS Patients with ACTH-dependent Cushing's syndrome (CS) patients were the subjects for this study. One blood sample each was collected from right and left IJV following intravenous hCRH at 3 and 5 min, respectively. A simultaneous peripheral vein sample was also collected with each IJV sample for calculation of IJV: Peripheral vein ACTH ratio. IJV sample collection was done under ultrasound guidance. ACTH was assayed using electrochemiluminescence immunoassay (ECLIA). RESULTS Thirty-two patients participated in this study. The IJV: Peripheral vein ACTH ratio ranged from 1.07 to 6.99 (n = 32). It was more than 1.6 in 23 patients. Cushing's disease could be confirmed in 20 of the 23 cases with IJV: Peripheral vein ratio more than 1.6. Four patients with Cushing's disease and 2 patients with ectopic ACTH syndrome had IJV: Peripheral vein ACTH ratio less than 1.6. Six cases with unknown ACTH source were excluded for calculation of sensitivity and specificity of the test. CONCLUSION IJV: Peripheral vein ACTH ratio calculated from a single sample from each IJV obtained after hCRH had 83% sensitivity and 100% specificity for diagnosis of CD.