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Background: Inferior petrosal sinus sampling (IPSS) is indicated in the diagnosis of adrenocorticotropic hormone (ACTH)-dependent Cushing's syndrome (CS), especially when the results of the initial diagnostic tests are discordant., Objective: To describe the patients who underwent this invasive functional test in a tertiary hospital., Methods: This was an observational study of a retrospective cohort of patients with ACTH-dependent CS and IPSS between 2004 and 2019. We determined their epidemiological, hormonal, radiological and functional characteristics, and evaluated their diagnostic capacity and optimal cut-off points to differentiate between Cushing's disease (CD) and ectopic Cushing's syndrome (ECS)., Results: 23 patients were evaluated, of which 65.2% were women with the average age of 42 (36-62) years. ACTH secretion of pituitary origin was evident in 82.6% of the patients and of ectopic origin in 17.4%. Plasma cortisol, urinary free cortisol, and ACTH levels were higher in patients with ECS. Regarding IPSS, the baseline central/peripheral ACTH gradient detected 89.5% of patients with CD and after stimulation with CRH, 100%. The optimal cut-off points in the diagnosis of CD were 2.06 at baseline and 2.49 after CRH stimulation., Conclusion: IPSS with CRH stimulation is a test with a high diagnostic accuracy for correctly classifying patients with CD and ECS. The cut-off points of the gradients may be different from the classic ones. Therefore, we recommend that each center perform its own evaluation., (Copyright © 2021 SEEN and SED. Published by Elsevier España, S.L.U. All rights reserved.)