Non-invasive imaging of transgene expression requires the appropriate combination of a reporter gene and a reporter probe. [¹⁸F]FEAU positron emission tomography (PET) is used for the assessment of herpes simplex virus type-1 thymidine kinase gene expression. Hybrid AAV phage (termed AAVP) can be adapted to transduce mammalian cells by targeting to a specific receptor. We evaluated a targeted AAVP vector using [¹⁸F]FEAU PET. This protocol describes [¹⁸F]FEAU production and dosing, micro-PET imaging and image analysis. 2-Deoxy-2-trifluoromethanesulfonyl-1,3,5-tri-O-benzoyl-α-D-ribofuranose is radio-fluorinated, converted into its 1-bromo derivative and coupled with protected 5-ethyl uracil. The coupled product is hydrolyzed and purified using HPLC. Tumor-bearing animals targeted with either retroviral or AAVP vectors are anesthetized and injected with [¹⁸F]FEAU (0.1 mCi per mouse); this is followed 2 h after injection by imaging on a micro-PET. Production of [¹⁸F]FEAU requires approximately 3.5 h from the end of bombardment. PET imaging studies require 2–3 h (depending on the number of animals) after synthesis of [¹⁸F]FEAU.