Open lung biopsy has proven beneficial in the treatment of life-threatening pulmonary diseases. Its safety and efficacy in infants and children placed on extracorporeal membrane oxygenation (ECMO) for severe respiratory failure is not known. The authors reviewed eight cases (4 neonates, 3 infants, 1 child) who underwent open lung biopsy while on ECMO. The primary diagnoses were pneumonia (4), lymphoma (1), primary pulmonary hypertension (1), and complex congenital heart disease (2). The patients underwent biopsy after they had been on ECMO an average of 9.6 days (range, 1 to 14 days). Biopsy results confirmed the clinical diagnosis in five patients, two of whom had irreversible alveolar destruction resulting in ECMO withdrawal. Three patients had pathological diagnoses, which resulted in major therapy revisions (1 fungal infection and 2 noninfectious lesions that required steroid treatment). The overall average duration of ECMO treatment was 16.3 days (range, 10 to 24 days). Three patients were weaned successfully from ECMO, but only one infant survived to discharge. One nonlethal bleeding complication occurred after biopsy. Open lung biopsy is well tolerated during ECMO. It accurately determines pulmonary pathology and provides valuable prognostic information. Earlier biopsy for patients whose diagnoses are uncertain or who are not responding to ECMO may improve the mortality rate for this high-risk group.