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Appropriate treatment of brainstem lesions is possible only when an accurate histopathologic diagnosis is made. Stereotactic biopsy has been used extensively to aid the diagnosis and management of intracranial lesions. This study presents the authors’ 18-year experience with diagnostic stereotactic biopsy of brainstem lesions.Hospital records for all patients with brainstem lesions who underwent stereotactic biopsy at our hospital between 1989 and 2007 were retrospectively reviewed.A stereotactic biopsy for brainstem tumors was performed on 200 patients (61% male) with a mean age of 23.9±16.97 years at the time of diagnosis. Glioma was the most common histopathologic diagnosis in 95.0% of the patients (n=190). Diagnoses in 10 patients included lymphoma (5), abscess (3), adenocarcinoma (1), and multiple sclerosis (1). The mortality was 0.5% and the morbidity was 4.0%: intraparenchymal hemorrhage, 5 cases; perilesional edema, 1 case; hydrocephalus, 1 case; and aspirative pneumonia, 1 case.Although the diverse pathology of brainstem lesions in adults requires an accurate diagnosis for the appropriate treatment strategy, the high incidence of brainstem glioma in children has led to some debate on the usefulness of the stereotactic biopsy in these patients.