Abstract BACKGROUND Accurate tissue sampling in nonenhancing (NE) gliomas is a unique surgical challenge due to their intratumoral histological heterogeneity and absence of contrast enhancement as a guide for intraoperative stereotactic guidance. Instead, T2/fluid-attenuated inversion-recovery (FLAIR) hyperintensity on MRI is commonly used as an imaging surrogate for pathological tissue, but sampling from this region can yield nondiagnostic or underdiagnostic brain tissue. Sodium fluorescein is an intraoperative fluorescent dye that has a high predictive value for tumor identification in areas of contrast enhancement and NE in glioblastomas. However, the underlying histopathological alterations in fluorescent regions of NE gliomas remain undefined. OBJECTIVE To evaluate whether fluorescein can identify diagnostic tissue and differentiate regions with higher malignant potential during surgery for NE gliomas, thus improving sampling accuracy. METHODS Thirteen patients who presented with NE, T2/FLAIR hyperintense lesions suspicious for glioma received fluorescein (10%, 3 mg/kg intravenously) during surgical resection. RESULTS Patchy fluorescence was identified within the T2/FLAIR hyperintense area in 10 of 13 (77%) patients. Samples taken from fluorescent regions were more likely to demonstrate diagnostic glioma tissue and cytologic atypia (P < .05). Fluorescein demonstrated a 95% positive predictive value for the presence of diagnostic tissue. Samples from areas of fluorescence also demonstrated greater total cell density and higher Ki-67 labeling than nonfluorescent biopsies (P < .05). CONCLUSION Fluorescence in NE gliomas is highly predictive of diagnostic tumor tissue and regions of higher cell density and proliferative activity.