Purpose: To develop a new glucuronide probe for micro-positron emission topography (PET) that can depict β-glucuronidase (βG)-expressing tumors in vivo. Materials and Methods: All animal experiments were preapproved by the Institutional Animal Care and Use Committee. A βG-specific probe was generated by labeling phenolphthalein glucuronide (PTH-G) with iodine 131 (131I) or 124I. To test the specificity of the probe in vitro, 124I-PTH-G was added to CT26 and βG-expressing CT26 (CT26/βG) cells. Mice bearing CT26 and CT26/βG tumors (n = 6) were injected with 124I-PTH-G and subjected to micro-PET imaging. A βG-specific inhibitor D-saccharic acid 1,4-lactone monohydrate was used in vitro and in vivo to ascertain the specificity of the glucuronide probes. Finally, the biodistributions of the probes were determined in selected organs after injection of 131I-PTH-G to mice bearing CT26 and CT26/βG tumors (n = 14). Differences in the radioactivity in CT26 and CT26/βG tumors were analyzed with the Wilcoxon signed rank test. Results: 124I-PTH-G was selectively converted to 124I-PTH (phenolphthalein), which accumulated in CT26/βG cells and tumors in vitro. The micro-PET images demonstrated enhanced activity in CT26/βG tumors resulting from βG-mediated conversion and trapping of the radioactive probes. Accumulation of radioactive signals was 3.6-, 3.4-, and 3.3-fold higher in the CT26/βG tumors than in parental CT26 tumors at 1, 3, and 20 hours, respectively, after injection of the probe (for all the three time points, P < .05). Conclusion: Hydrophilic-hydrophobic conversion of 124I-PTH-G probe can aid in imaging of βG-expressing tumors in vivo.