Context: Pituitary adenomas in CD are usually small and difficult to visualize. Bilateral inferior petrosal venous sampling (BIPSS) before and after ovine (o) CRH (CRH) stimulation is reserved for patients who have corticotropin (ACTH)-dependent CS and negative magnetic resonance imaging (MRI), or positive MRI but inconsistent biochemical data. Objective: To evaluate the usefulness of BIPSS as a tool for localization of a pituitary adenoma in children with CD. Design: Retrospective review of the records of 141 children who were admitted for evaluation of CD from 1982 - 2004. Setting: Tertiary care center. Interventions and outcome measures: Lateralization of ACTH secretion during BIPSS was compared with MRI and surgical findings for the localization of a microadenoma. Results: A total of 94 patients, 49 males and 45 females with an age range of 5.3 to 18.7 yr (13 ± 3.2 yr), underwent BIPSS. Localization of a microadenoma by BIPSS agreed with surgical location in only 58% of the cases (CI₉₅= 43 - 66). The combined use of information from the MRI and the IPSS did not predict the location of the tumor more frequently than MRI alone (P > 0.1), which in this study localized a lesion in 39% of the patients (CI₉₅= 28 - 50). The procedure was completed successfully in all patients and no serious complications were recorded. Conclusions: Although BIPSS was safe and well tolerated in an experienced center, lateralization of the ACTH gradient during BIPSS was a poor predictor of the site of the adenoma in children with CD.