We aimed to determine the effect of carbon ion irradiation on the uptake of N-benzyl-N-11C-methyl-2-(7-methyl-8-oxo-2-phenyl-7,8-dihydro-9H-purin-9-yl)acetamide ([(11)C]DAC), a positron emission tomography (PET) ligand for the peripheral-type benzodiazepine receptor (PBR), in tumor cells and tumor-bearing mice. Spontaneous murine fibrosarcoma (NFSa) cells were implanted into the right hind legs of syngeneic C3H male mice. Conditioning irradiation with 290 MeV/u carbon ions was delivered to the 7- to 8-mm tumors In vitro uptake of [(11)C]DAC was measured in single NFSa cells isolated from NFSa-bearing mice after irradiation. In vivo biodistribution of [(11)C]DAC in NFSa-bearing mice was determined by small animal PET scanning and dissection. In vitro autoradiography was performed using tumor sections prepared from mice after PET scanning. In vitro and in vivo uptake of [(11)C]DAC in single NFSa cells and NFSa-bearing mice was significantly reduced by carbon ion irradiation. The decrease in [(11)C]DAC uptake in the tumor sections was mainly due to the change in PBR expression. In conclusion, [(11)C]DAC PET responded to the change in PBR expression in tumors caused by carbon ion irradiation in this study. Thus, [(11)C]DAC is a promising predictor for evaluating the effect of carbon ion radiotherapy.