Because of the increasing application of stereotactic neurosurgical techniques not only in the diagnosis but also in the treatment of brain tumors, a demand for data regarding the spatial delimitation of gliomas is being created. Traditional histologic criteria were not established for detecting isolated tumor cells located at the boundaries of gliomas. Standard histologic techniques used for processing surgical specimens result in preparations of insufficient quality to allow the detection of minimal tumor infiltrates within normal brain parenchyma. Consequently, histologic examination has been considered an unreliable method of determining the boundaries of gliomas. In this study, we describe in detail an improved technique of biopsy specimen preparation that involves the use of glutaraldehyde-fixed and hemalum-phloxine-stained paraffin sections as well as alcohol-fixed and hemalum-phloxine-stained smears. In concert, these methods allow visualization of delicate cytologic details and are complementary in assessing the presence of isolated tumor cells at the periphery of gliomas. We define morphologic criteria for the accurate identification of isolated neoplastic cells and for their distinction from normal or reactive glial elements. These morphologic criteria include classic cytologic features of malignant lesions (such as nuclear pleomorphism and hyperchromasia) as well as the assessment of tumor architecture and cellular spatial relationships.