In a rat AH109A tumor model, metabolic tracers for glucose, amino acid, and nucleic acid metabolisms (2-deoxy-2-[18F]fluoro-D-glucose (18FDG), L-[methyl-14C]methionine (14C-Met), [6-3H]thymidine (3H-Thd), and 2'-deoxy-5-[18F]fluorouridine (18FdUrd], and the conventional radionuclide 67Ga-citrate were used to assess the feasibility of monitoring tumor radiotherapy using a quadruple tracer technique. Two combinations of four tracers (18FDG or 18FdUrd, 14C-Met, 3H-Thd and 67Ga) were compared in a time-course study after single-dose irradiation (20 Gy) and were also used in a dose-dependency study performed 6 days after 5, 10, 15, or 20 Gy of irradiation. Fluorine-18-FDG showed a large change in uptake and a steady response to radiotherapy. Fluorodeoxyuridine showed a rapid decrease after radiotherapy, but the range of change in uptake was narrow. Gallium-67 could not detect tumor response early after treatment, but showed a marked change in uptake later. [6-3H]Thd and 14C-Met showed a rapid response to irradiation and a high sensitivity for monitoring radiotherapy, suggesting that they may be feasible for PET studies.