The aim of this study was to develop an automated synthesis of 9-(4-[ 18 F]-fluoro-3-hydroxymethylbutyl)guanine ([ 18 F]FHBG) and 9-[(3-[ 18 F]fluoro-1-hydroxy-2-propoxy)methyl]guanine ([ 18 F]FHPG) using a Scanditronix Anatech RB III robotic system. [ 18 F]HF was produced via 18 O(p, n) 18 F using a Scanditronix MC17F cyclotron. On average, a typical run produced [ 18 F]FHBG and [ 18 F]FHPG with an uncorrected radiochemical yield of 19% and 16%, respectively, at end of synthesis (EOS) from irradiation of 95% enriched [ 18 O]water. The total synthesis time was 80 min. The retention time of [ 18 F]FHBG and [ 18 F]FHPG (the radio-peak) was 3.9 and 4.0min, respectively, which was consistent with the [ 19 F]FHBG and [ 19 F]FHPG ultraviolet peak. The radiochemical purity was greater than 97%. A robotic, automated method for [ 18 F]FHBG and [ 18 F]FHPG radiosynthesis is therefore feasible. The radiation burden for the operator can be reduced as much as possible. Sufficient radioactivities of [ 18 F]FHBG and [ 18 F]FHPG could be obtained for non-invasive monitoring the expression of transfected gene in vivo with positron emission tomography (PET).