Abstract: 3’-Deoxy-3’-¹⁸Ffluorothymidine (¹⁸F-FLT) is a radiotracer for the imaging of tumor proliferation. A clinically applicable automatic system for the preparation of ¹⁸F-FLT was developed by modifying a domestic ¹⁸F-FDG synthesizer with semipreparative HPLC. Fifteen milligrams of 3-N-Boc-5’-O-dimethoxytrityl-3’-O-nosyl-thymidine were dissolved in 0.5 mL DMSO and reacted with dried ¹⁸F-fluoride at 100 ℃ for 5 min. The obtained material was hydrolyzed with 1 mol/L HCl at 110 ℃ for 5 min, and then neutralized with 2 mol/L NaOH before HPLC purification was performed. The desired radioactive fraction was collected after passing through a 0.22 m filter into a 30 mL vial as the final product. The ¹⁸F-FLT labelling yield was found to be 67.5% (n=8) by the radio-TLC method, and 39.4% (n=6) by the HPLC method. The yield as the final product for clinical use was 21.2% (n=3, not corrected for decay). The total preparation time, including the time for HPLC purification, was 30 min. The radiochemical purity of the final product was over 99%, and the specific activity was higher than 740 TBq/mg (180 PBq/mol). The final product was stable for more than 6 h in the 10% alcohol solution. This preparation system with semipreparative HPLC enables us to produce ¹⁸F-FLT with a stable yield for clinical use.