Abstract Purpose The response of 2-amino-4-([ 14C]methylthio)butyric acid ([ 14C]Met) uptake and [ 125I]3-iodo-alpha-methyl- l-tyrosine ([ 125I]IMT) uptake to radiotherapy of C10 glioma cells was compared to elucidate the intracellular reactions that affect the response of 2-amino-4-([ 11C]methylthio)butyric acid ([ 11C]Met) uptake to radiotherapy. Methods After irradiation of cultured (3 Gy) or xenografted C10 glioma cells (25 Gy) using a carbon ion beam, the accumulation of [ 14C]Met and [ 125I]IMT in the tumors was investigated. The radiometabolites in xenografted tumors after radiotherapy were analyzed by size-exclusion HPLC. Results [ 14C]Met provided earlier responses to the carbon ion beam irradiation than [ 125I]IMT in both cultured and xenografted tumors. While [ 125I]IMT remained intact in xenografted tumor before and after irradiation, the radioactivity derived from [ 14C]Met was observed both in high molecular fractions and intact fractions, and the former decreased after irradiation. Conclusion The earlier response of [ 11C]Met uptake to tumor radiotherapy could be attributable to the decline in the intracellular energy-dependent reactions of tumors due to radiotherapy.