© 2021 Elsevier B.V.Introduction: Bilateral inferior petrosal sinus sampling (BIPSS) is an important procedure in the diagnostic work-up of Cushing's syndrome (CS). In this study, we investigated the diagnostic performance of BIPSS in detecting the source of adrenocorticotropic hormone (ACTH) secretion in Cushing's disease (CD) without radiological evidence. Methods: Thirty-five consecutive patients who underwent BIPSS due to ACTH-dependent CS between 2010 and 2019 in our clinic and subsequently underwent transsphenoidal surgery were included. The indication for BIPSS was biochemically proven ACTH-dependent CS but normal or ≤6 mm pituitary lesion in pituitary magnetic resonance imaging (MRI). Corticotropin releasing hormone (CRH) stimulation was applied to all patients during the BIPSS procedure. BIPSS data, MRI results, pathological findings, and follow-up results were analyzed. The diagnostic performance of BIPSS was calculated. Results: A total of 35 patients, 6 (17%) males and 29 (83%) females, were included in the study. Pituitary MRI was normal in 12 (34.3%) and revealed lesions ≤ 6 mm in 23 (65.7%) patients. BIPSS lateralized the right side in 13 (37.1%) and left side in 18 (51.4%) patients, while no lateralization was observed in the remaining 4 (11.5%) patients. BIPSS showed lateralization in the same direction with pituitary adenoma in 21 (60%) patients before CRH injection and in 29 (83%) patients after CRH injection (p = 0.034). The sensitivity of the BIPSS procedure was 88%. Accurate localization of the pituitary lesion was more frequent when based on BIPSS results than on MRI (83% vs. 51%, P = 0.005). Conclusion: BIPSS appears to be a valuable and safe diagnostic tool in patients who are diagnosed with CD biochemically but do not have clear radiological evidence of ACTH-producing lesion.