Beta-glucuronidase (beta G) is a potential biomarker for cancer diagnosis and prodrug therapy. The ability to image beta G activity in patients would assist in personalized glucuronide prodrug cancer therapy. However, whole-body imaging of beta G activity for medical usage is not yet available. Here, we developed a radioactive beta G activity-based trapping probe for positron emission tomography (PET). We generated a I-124-tyramine-conjugated difluoromethylphenol beta-glucuronide probe (TrapG) to form I-124-TrapG that could be selectively activated by bG for subsequent attachment of I-124-tyramine to nucleophilic moieties near beta G-expressing sites. We estimated the specificity of a fluorescent FITC-TrapG, the cytotoxicity of tyramine-TrapG, and the serum half-life of I-124-TrapG. beta G targeting of I-124-TrapG in vivo was examined by micro-PET. The biodistribution of I-131-TrapG was investigated in different organs. Finally, we imaged the endogenous bG activity and assessed its correlation with therapeutic efficacy of 9-aminocamptothecin glucuronide (9ACG) prodrug in native tumors. FITC-TrapG showed specific trapping at beta G-expressing CT26 (CT26/m beta G) cells but not in CT26 cells. The native TrapG probe possessed low cytotoxicity. I-124-TrapG preferentially accumulated in CT26/m beta G but not CT26 cells. Meanwhile, micro-PET and whole-body autoradiography results demonstrated that I-124-TrapG signals in CT26/m beta G tumors were 141.4-fold greater than in CT26 tumors. Importantly, Colo205 xenografts in nude mice that express elevated endogenous beta G can be monitored by using infrared glucuronide trapping probes (NIR-TrapG) and suppressed by 9ACG prodrug treatment. I-124-TrapG exhibited low cytotoxicity allowing long-term monitoring of bG activity in vivo to aid in the optimization of prodrug targeted therapy. (C) 2014 AACR.