Objective: The aim of this study was to determine the accuracy of bilateral cavernous sinus sampling (CSS) in preoperative tumor lateralization (right/left) within the pituitary in patients with Cushing's disease (CD). Patients and methods: The study consisted of 26 consecutive patients who had undergone CSS followed by transsphenoidal surgery (TS) for CD between 2000 and 2006 at our institution. The magnetic resonance imaging (MRI) of the selected patients either revealed a normal pituitary or a lesion ≤6 mm within the gland. Simultaneous bilateral CSS with corticotropin releasing hormone (CRH) stimulation were performed in all cases and the data was analyzed in relation to the results of the MRI studies, intraoperative and pathological findings and the outcome. Results: Early remission was achieved in 23 patients (88%) and CSS predicted the correct localization of the adenoma in 22 patients (85%). No lateralization (elevated levels in both sides) was detected during CSS in two patients, due to lesions within the central part of the pituitary. In four cases, there was a false positive lateralization, in which no microadenoma could be located in the lateralized side of the pituitary, resulting in no remission. There were no complications related to the CSS. Conclusion: CSS in CD seems to be a valuable and safe diagnostic tool, which can predict the correct location of the pituitary adenoma in 85% of the cases. © 2008.