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To analyze the bilateral and simultaneous petrosal sinus sampling (BIPSS) in a subgroup of children and adolescents with ACTH-dependent Cushing's syndrome (ADCS) METHODS: Retrospective study in a tertiary reference center. From 1993 and 2017, 19 children and adolescents (PED) were submitted to the BIPSS, median age of 14 years (range 9-19 years), 53% were males, 18 had Cushing's disease (CD) and one had ectopic ACTH syndrome (EAS). All procedures were performed with 10 µg of intravenous desmopressin.The catheter positioning was successful in all cases. The central ACTH gradient was met in 17/19 cases. At baseline, central gradient occurred in 16/19 (84%) with gradient values of 7.2 ± 6.0. After stimulation, there was an increase in the center-periphery gradient values (33.6 ± 44.3). In one case, central gradient was defined only after stimulation. Two cases presented without a central gradient; one case of CD with a false-negative and one EAS case. Lateralization occurred in all cases with a central gradient. Confirmation of the tumor location presumed by the procedure with the surgical description occurred in 60% of the cases. The BIPSS in this PED subgroup of ADCS presented a sensitivity of 94.4% and specificity of 100%. There were no complications of the procedure.In a series of children and adolescents with ADCS, BIPSS was safe and highly accurate in defining the central to peripheral ACTH gradient using desmopressin as secretagogue. Nevertheless, there was a limited value of the ACTH-gradient between the petrosal sinuses for the tumor location.