BACKGROUND: The radiolabeled amino acid O-(2-18F-fluoroethyl)-L-tyrosine (18F-FET) and thymidine analog 3'-deoxy-3'-18F-fluorothimidine (18F-FLT) are widely used for positron emission tomography (PET) brain tumor imaging; however, results from comparative studies are scarce. Due to the limited transport of FLT across the intact blood-brain barrier (BBB) we hypothesize that anti-VEGF response assessment with FLT PET primarily reflects changes in the BBB, whereas FET PET is less affected by the tumor vasculature. The aim of this study was therefore to evaluate and compare the value of FLT and FET MicroPET for anti-VEGF response assessment in orthotopic human glioma xenografts. METHODS: Cells of human glioblasoma multiforme (GBM) neurosphere culture (NGBM_CPH048p6_LUC) were injected orthotopically in NMRI nude mice. At confirmed tumor take, mice were treated with anti-VEGF therapy (B20-4.1) or saline as control. Treatment response was followed by weekly bioluminescence, 18F-FLT and 18F-FET MicroPET/CT. The end-point was survival and brains from sacrificed mice were used for immunohistochemistry and subsequent quantification of the Ki67 proliferation index and micro-vessel density (MVD). RESULTS: The relative 18F-FET tumor-to-brain of (T/B) ratio of SUVmax was significantly decreased after one week (99 ± 6%, n = 6 vs. 124 ± 9%, n = 5; p = 0.04) and after two weeks (106 ± 10%, n = 5 vs. 156 ± 17%, n = 4; p = 0.03) in the B20-4.1 group as compared with the control group. However, with 18F-FLT MicroPET there was no significant difference in the T/B ratio of SUVmax neither after one week of treatment (134 ± 12%, n = 6 vs. 144 ± 6%, n = 6; p = 0.49) nor after two weeks of treatment (174 ± 11%, n = 5 vs. 208 ± 30%, n = 5; p = 0.34). We found a significant lower MVD in the B20-4.1 group as compared to the control group (66 ± 8%, n = 8 vs. 100 ± 10%, n = 7; p = 0.02). However, we found no difference in the Ki67 proliferation index in the treatment group as compared to the control group (18.9 ± 1%, n = 7 vs. 19.8 ± 2%; n = 6; p = 0.7). In addition, the median survival was not prolonged in the treatment group as compared to the control group (19 vs. 21 days, p = 0.34). CONCLUSION: In NGBM_CPH048p6_LUC xenografts B20-4.1 anti-VEGF treatment induced changes in the MVD and in 18F-FET uptake without affecting the Ki67 proliferation index. Surprisingly and in contradiction with the hypothesis, the 18F-FLT uptake was not affected by the changes in the MVD.