The ACTH-producing microadenoma is by far the most common cause of hypercortisolism in patients with Cushing's syndrome. Selective transsphenoidal surgery is the treatment of choice for Cushing's disease, however, it is not uniformly successful. Aim of this study was to determine simultaneously the ACTH-concentration in the right and left sinus petrosus inferior in patients with Cushing's syndrome before and after stimulation with CRF. The procedure was attempted in 17, successfully carried out in 16 patients. In the 14 of whom Cushing's disease was diagnosed, 11 had a gradient greater than 1.4 (central versus peripheral ACTH) after administration of CRF, two patients had no gradient, yet responded to CRF. One patient had the ectopic ACTH-syndrome and showed no response to CRF and no gradient. One patient, found to have no Cushing's disease, had no gradient and responded normal to CRF. In 9 of the 11 patients with a gradient, the microadenoma was found on the side of the higher ACTH-level; one patient underwent hemihypophysectomy of the side where the gradient was directed towards and was cured. No complications occurred. We conclude, that selective, simultaneous and bilateral catheterization of the sinus petrosus inferior is a safe method to localize the side of the microadenoma preoperatively.