Background: Differential diagnosis of the etiology of ACTH-dependent hypercortisolism is very difficult in some cases. The uncertainty, whether there is a central or ectopic etiology, is also strengthened by the fact that even nuclear magnetic resonance (MR) sometimes does not prove the pituitary adenoma. A more precise differential diagnosis under these conditions has been made, over the last years, by selective determination of ACTH levels bilaterally in sinus petrosi inferiores (SPI) and by their comparison with the level in peripheral blood. The confirmation of central etiology makes a decision about neurosurgical intervention easier. This intervention, due to the present microsurgery technique, makes it possible to detect otherwise undetectable microadenoma and enables its selective excision with maintaining healthy hypophysis tissue and all its functions., Methods and Results: In the last two years the authors indicated 11 patients (10 women, one man at the age of 17-54 years, mean age 33.5 year) with proved ACTH-dependent hypercortisolism to the determination of ACTH in SPI. Their CT or NMR examination did not prove a hypophysis tumour unequivocally. Only in one female patient the catheterization was not possible for anomalous vascular bed with aplasia of internal jugular vein on the left and hypoplasia of internal jugular vein on the right. In 8 patients the determination of the ratio of ACTH levels between SPI and periphery (7.78 +/- 1.54, mean +/- S.E.) has shown that even the basal levels indicated central etiology of hypercortisolism. This has been confirmed by the ratio of the values of ACTH after stimulation with corticotrophin releasing hormone (CRH). The diagnosis was confirmed in all patients by surgery, by histology and post-operation course. In two female patients the ratio of basal levels of ACTH, SPI/periphery did not prove useful for the diagnosis of the Cushing's disease. In the first patient the CRH stimulation was not yet performed routinely and the ratio of basal levels of ACTH, SPI/periphery gave a false negative result, as shown by the finding of adenoma of hypophysis during the operation. The operation was indicated on the basis of hormonal examinations and repeated MR with a finding of suspected microadenoma and the diagnosis was then confirmed by further clinical development. The determination of the levels of ACTH after CRH stimulation would give more precision in this case, as shown by analysis of the results of large cohorts in literature, which we perform at present as well. In the other patient the CRH stimulation test was done, changing the ratio from 1.1 to 5.7 and thus confirmed central etiology of the Cushing's syndrome. In agreement with literature this method did not contribute to lateral localization of the tumour., Conclusions: The determination of basal levels of ACTH in SPI and levels after CRH stimulation enables to differentiate between central and ectopic overproduction of ACTH in the ACTH-dependent Cushing's syndrome.