The mediastinum is the most common site for primary intra-thoracic pathology in childhood. This paper describes the versatility of the Chamberlain operation in establishing tissue diagnosis and guiding definitive treatment in children with mediastinal mass lesions. During 1999-2006, 28 consecutive patients were referred to a National Oncology Center with anterior mediastinal pathology. Eleven underwent the Chamberlain procedure. Demographic data, diagnostic sensitivity, operating time and morbidity were recorded. All patients had preoperative chest X-ray and computed tomography scans. The Chamberlain operation was deployed in only those children without other ways of accurately establishing their diagnosis. Others with mediastinal pathology had a diagnosis established by lymph node biopsy, thoracentesis or other method(s). Eleven patients (nine male, two female; age range 2-13 years) underwent the Chamberlain procedure. In these children, there was no pre-existent diagnosis and this was the primary procedure employed. Diagnostic accuracy was 100%. Three patients had pleural disruption and chest tubes were placed at the time of surgery. No patient required a thoracotomy. Average operating time was 1.3 h. Five patients were diagnosed with Hodgkin's lymphoma, four had non-Hodgkin's lymphoma and two children thymic hyperplasia. The Chamberlain operation provides excellent access to the antero-superior mediastinum for biopsy of obscure mediastinal mass lesions in childhood. Complications from this procedure are very rare.