Objective: The uptake of 3′-[18F]fluoro-3′- deoxythymidine (FLT), a proliferation marker, was measured before and during fractionated radiotherapy to evaluate the potential of FLT-positron emission tomography (PET) imaging as an indicator of tumor response compared to 2′-deoxy-2′-[18F]fluoro-d-glucose (FDG). Materials and Methods: Nude mice bearing established human head and neck xenografts (HNX-OE; nu/nu mice) were locally irradiated (three fractions/week; 22 Gy) using a 150-kVp unit. Multiple FDG- and FLT-PET scans were acquired during treatment. Tumor volume was determined regularly, and tissue was analyzed for biomarkers involved in tracer uptake. Results: Both groups revealed a significant decline in tumor volume (P∈<∈0.01) compared to untreated tumors. For FDG as well as for FLT, a significant decline in retention was observed at day 4. For FLT, most significant decline in retention was observed at day 12; whereas, for FDG, this was already noted at day 4. Maximum decline in tumor-to-nontumor ratios (T/NT) for FDG and FLT was 42∈±∈18% and 49∈±∈16% (mean∈± ∈SD), respectively. FLT uptake was higher then that of FDG. For FLT, statistical significant correlations were found for both tumor volume at baseline and at day 29 with T/NT and ΔT/NT. All tumors demonstrated expression of glucose transporter-1, thymidine kinase-1, and hexokinase II. No differences were found for amount of tumor cells and necrosis at the end of treatment. Conclusion: This new experimental in vivo model supports the promise of using FLT-PET, as with FDG-PET, to monitor response to external radiotherapy. This warrants further clinical studies to compare these two tracers especially in cancers treated with radiotherapy.