OBJECTIVE As an alternative method to inferior petrosal sinus sampling with administration of corticotropin-releasing hormone (CRH), the authors have developed a method of selective venous sampling directly from the cavernous sinus, which is performed using a superselective catheterization technique. The goal of this study is to assess this method. METHODS Catheterization to the cavernous sinus was performed in 44 patients with clinical and biochemical features of Cushing's syndrome. Forty of these patients in whom there was a definite clinical and/or histological diagnosis were evaluated. In 35 patients, a pituitary microadenoma was detected on magnetic resonance imaging and/or during surgery. Ectopic lesions were suspected in five patients, who later developed nonpituitary tumors. The central/peripheral (C/P) ratios of adrenocorticotrophic hormone (ACTH) without CRH administration ranged from 5.2 to 448.1 (mean+/-standard deviation, 66.7+/-91.2) in cavernous sinuses and from 1.1 to 52 (mean+/-standard deviation, 8.5+/-9.9) in the inferior petrosal sinuses. The petrosal sinus sampling contained false negative results in eight patients (23%) based on a cutoff point of 2. On the contrary, patients with ectopic lesions did not show any increase in ACTH levels in either sampling portion. The intercavernous gradients of ACTH, ranging from 1.2 to 506.4, indicated the correct lateralization of microadenoma in the lateral wing in all 30 patients. The interpetrosal gradients, ranging from 1 to 31.4, did not give a sufficient value (> or = 1.4) in four patients (13%) with a lateral lesion. In five cases of midline tumors, the intercavernous and interpetrosal gradients indicated false laterality in three and four cases, respectively. The intracavernous (posterior-anterior) gradients, ranging from 1.04 to 60.7 (mean 14.9), showed a higher concentration of ACTH in the posterior portion of the sinus in all patients. CONCLUSIONS These results suggest that 1) cavernous sinus sampling without CRH administration can demonstrate hypersecretion of ACTH from the pituitary gland with a high diagnostic accuracy; 2) intercavernous gradients will indicate the correct lateralization in laterally localized microadenomas; and 3) sampling should be performed from the posterior portion of the cavernous sinus.