Detection of anaplastic tumor foci for precise grading of gliomas is crucial for prognostic assessment and appropriate postoperative treatment planning. To avoided undergrading in large suspected low-grade gliomas, we employed frameless sterotaxy during open surgery for tissue sampling of radiologically suspected anaplastic foci. In nine patients (mean age 44 years, range 10-67) with large supratentorial suspected low-grade gliomas (32.4 ccm mean vol, range 17.9-68.6 ccm) with small contrast enhancing areas (7.7% mean of total volume, range 0.7-15.3%), a neuronavigation system with a pointer device (Easy Guide System Philips) or a navigating microscope (MKM System Zeiss) was used to target small enhancing tumor parts for cytological investigation during open surgery. Consecutive cytological smears revealed anaplastic tumor foci in all patients, correlating with neuroradiologically demonstrated small contrast enhancing areas, although biopsies from all other tumor parts showed low-grade tumors. Final neuropathological diagnosis confirmed anaplasia within the neuroradiologically suspected low-grade gliomas in all patients (2 grade IV, 5 WHO grade III and 2 grade II-III tumors, WHO classification). In our experience frameless stereotactic-directed intraoperative tissue sampling during open surgery of large suspected low-grade gliomas helps to identify small anaplastic areas in mostly large low-grade tumors, and therefore, permits optimum planning of postoperative treatment.