The aim of this study was to compare preoperative dual-time point F-fluorodeoxyglucose (FDG) uptake pattern with intraoperative 5-aminolevulinic acid (5-ALA) fluorescence in high-grade gliomas. In addition, we assessed for possible associations with a pathologic parameter (Ki-67 index).Thirty-one patients with high-grade glioma (M:F = 19:12, mean age = 60.6 ± 11.2 years) who underwent dual-time point F-FDG positron emission tomography (PET)/computed tomography (CT) scan before surgery were retrospectively enrolled; 5-ALA was applied to the surgical field of all these patients and its fluorescence intensity was evaluated during surgery. Measured F-FDG PET/CT parameters were maximum and peak tumor-to-background ratio (maxTBR and peakTBR) at base (-base) and delayed (-delay) scan. The intensity of 5-ALA fluorescence was graded on a scale of three (grade I as no or mild intensity, grade II as moderate intensity, and grade III as strong intensity).Seven of the patients had WHO grade III brain tumors and 24 had WHO grade IV tumors (mean tumor size = 4.8 ± 1.8 cm). MaxTBR-delay and peakTBR-delay showed significantly higher values than maxTBR-base and peakTBR-base, respectively (all P < .001). Among the F-FDG PET/CT parameters, only maxTBR-delay demonstrated significance according to grade of 5-ALA (P = .030), and maxTBR-delay gradually decreased as the fluorescence intensity increased. Also, maxTBR-delay and peakTBR-delay showed significant positive correlation with Ki-67 index (P = .011 and .009, respectively).Delayed F-FDG uptake on PET/CT images could reflect proliferation in high-grade glioma, and it has a complementary role with 5-ALA fluorescence.