TM-601, a 36-amino-acid peptide, selectively binds to glioma cells but not normal brain parenchyma. A phase I/II clinical trial of intracavitary 131I-TM-601 in adult patients with recurrent high-grade glioma was performed to determine the biodistribution and toxicity of this potential therapy. We evaluated imaging and biodistribution data from this trial to assess whether 131I-TM-601 might be useful in determining tumor extent. Methods: Adult patients with recurrent high-grade gliomas underwent tumor resection, implantation of an intracavitary reservoir, and a single-dose injection of 370 MBq (10 mCi) 131I-TM-G01 (0.25-1.0 mg of 131I-TM-601) 2-4 wks after surgery. Total-body planar scans and whole-brain SPECT scans were obtained on days 0, 1, 2, 3, and 6-8 after injection. Postresection MR images were coregistered to the SPECT scans using image analysis software. Analysis of the rate of radioactive decay and biologic elimination from the body and at the cavity site was performed. T1-weighted with gadolinium contrast (T1-Wc), T2-weighted (T2), and SPECT volumes were estimated by stereological Cavalieri sections and compared for overlap. Results: Nonbound 131I-TM-601 was eliminated by 48 h after injection with the remaining radiolabeled peptide bound to tumor for at least 6-8 d. Biologic decay rates from 24 to 168 h after injection were only slightly shorter than the physical decay of 131I (6.3 vs. 8.0 d). A comparison of tumor volume estimates using all 3 imaging parameters indicated that 131I-TM-601- determined tumor volumes more closely paralleled T2 volumes than T1-Wc volumes. Overlap between coregistered MRI and SPECT scans corroborated the presence of radiolabeled peptide in the vicinity of infiltrating tumor up to 168 h after injection. Conclusion: 131I-TM-601 provides a reliable estimate for primary tumor extent. Further modification of this radiopeptide with other better imaging isotopes may provide an important tool for determining tumor extent and differentiating regions of viable tumor from necrosis.