Introduction: [18F]-Labeled analogues of thymidine have demonstrated efficacy for PET imaging of cellular proliferation. We have synthesized two [18F]-labeled N3-substituted thymidine analogues, N3-[18F]fluoroethyl thymidine (N3-[18F]-FET) and N3-[18F]fluoropropyl thymidine (N3-[18F]-FPrT), and performed preliminary PET imaging studies in tumor-bearing mice. Methods: Thymidine was converted to its 3′,5′-O-bis-tetrahydropyranyl ether, which was then converted to the N3-ethyl and propyl-substituted mesylate precursors. Reactions of these mesylate precursors with n-Bu4N[18F] or K[18F]/kryptofix followed by acid hydrolysis and HPLC purification yielded N3-[18F]-FET and N3-[18F]-FPrT, respectively. Subcutaneous (sc) xenografts of H441 human non-small cell lung cancer were established in two groups of mice (each n=6). Micro-PET images of the tumor-bearing animals were acquired after intravenous injection of N3-[18F]-FET or N3-[18F]-FPrT (3700 KBq/animal). Results: The radiochemical yields were 2-12% (d.c.) for N3-[18F]-FET and 30-38% (d.c.) for N3-[18F]-FPrT. Radiochemical purity was >99% and calculated specific activity was >74 GBq/μmol at the end of synthesis. The accumulation of N3-[18F]-FET and N3-[18F]-FPrT in the tumor tissue at 2 h postinjection was 1.81±0.78 and 2.95±1.14 percent injected dose per gram (%ID/g), respectively; tumor/muscle ratios were 5.57±0.82 and 7.69±2.18, respectively; the unidirectional influx rates (Ki) were 0.013 and 0.018 ml/g per minute, respectively. Conclusion: Two novel [18F]- N3-substituted thymidine analogues have been synthesized in good yields, high purity and high specific activity. Preliminary in vivo studies demonstrated the efficacy of these [18F]- N3-substituted thymidine analogues for PET imaging of tumors.