Purpose: Several radioisotope-labeled thymidine derivatives such as [¹¹C]thymidine was developed to demonstrate cell proliferation in tumor. But it is difficult to track metabolism with [¹¹C]thymidine due to rapid in vivo degradation and its short physical half-life. 3'-[¹⁸F]fluoro-3'-deoxythymidine ([¹⁸F]FLT) was reported to have the longer half life of fluorine-18 and the lack of metabolic degradation in vivo. Here, we described the synthesis of the 3´-[¹⁸F]fluoro-3´-deoxythymidine ([¹⁸F]FLT) and compared with [¹⁸F]FET and [¹⁸F]FDG in cultured 9L cell and obtained the biodistribution and PET image in 9L tumor bearing rats. Material and Methods: For the synthesis of [¹⁸F]FLT, 3-N-tert-butoxycarbonyl-(5'-O-(4,4'-dimethoxytriphenylmethyl)-2'-deoxy-3'-O-(4-nitrobenzenesulfonyl)-β-D-threopentofura nosyl)thymine was used as a FLT precursor, on which the tert-butyloxycarbonyl group was introduced to protect N3-position and nitrobenzenesulfonyl group. Radiolabeling of nosyl substitued precursor with 18F was performed in acetonitrile at 120℃ and deproteced with 0.5 N HCl. The cell uptake was measured in cultured 9L glioma cell. The biodistribution was evaluated in 9L tumor bearing rats after intravenous injection at 10 min, 30 min, 60 min and 120 min and obtained PET image 60 minutes after injection. Results: The radiochemical yield was about 20-30% and radiochemical purity was more than 95% after HPLC purification. Cellular uptake of [¹⁸F]FLT was increased as time elapsed. At 120 min post-injection, the ratios of tumor/blood, tumor/muscle and tumor/brain were 1.61±0.34, 1.70 ±0.30 and 9.33±2.22, respectively. The 9L tumor was well visualized at 60 min post injection in PET image. Conclusion: The uptake of [¹⁸F]FLT in tumor was higher than in normal brain and PET image of [¹⁸F]FLT was acceptable. These results suggest the possibility of [¹⁸F]FLT as an imaging agent for brain tumor.(Nucl Med Mol Imaging 2006;40(5):263-270)