This animal study investigates the application of positron emission tomography (PET) with tracers of tumour metabolism for monitoring suicide gene therapy with herpes simplex virus thymidine kinase (HSVtk). After transplantation of HSVtk-expressing Morris hepatoma cells into ACI rats, dynamic PET measurements of 18F-labeled 2-fluoro-2-deoxyglucose (FDG) uptake were performed in animals 2 days (n = 7) and 4 days (n = 5) after the onset of therapy with 100 mg ganciclovir (GCV)/kg body weight as well as after administration of sodium chloride (n = 8). The arterial FDG plasma concentration was measured dynamically in an extracorporeal loop and the rate constants for FDG transport (K1, k2) and FDG phosphorylation (k3) were calculated using a three-compartment model modified for heterogeneous tissues. Also, quantification using the metabolic rate of FDG turnover and the standardized uptake value (SUV) was done. Furthermore, the thymidine incorporation into the tumour DNA was determined after i.v. administration of 3H-thymidine. An uncoupling of FDG transport and phosphorylation was found with enhanced K1 and k2 values and a normal k3 after 2 days of GCV treatment. The increase in FDG transport normalized after 4 days whereas the phosphorylation rate k3 increased. Quantification using the metabolic rate or the SUV showed congruent but less sensitive results compared with the modeling approach. The thymidine incorporation into the DNA of the tumours declined to 10.5% of the controls after 4 days of GCV treatment. The data indicate that PET with 18FDG and 11C-thymidine may be applied for monitoring of gene therapy with the HSVtk/GCV suicide system. Increased transport rates are evidence of stress reactions early after therapy. The measurement of thymidine incorporation into the tumour DNA can be used as an indicator of therapy efficacy.