Quantitative 39-deoxy-39-18 F-fluorothymidine ( 18 F-FLT) PET has potential as a noninvasive tumor biomarker for the objective assessment of response to treatment. To guide interpretation of these quantitative data, we evaluated the repeatability of 18 F-FLT PET as part of a multicenter trial involving patients with high-grade glioma. Methods: 18 F-FLT PET was performed on 10 patients with recurrent high-grade glioma at 5 different institutions within the Adult Brain Tumor Consortium trial ABTC1101. Data were acquired according to a double baseline protocol in which PET examinations were repeated within 2 d of each other with no intervening treatment. On each of the 2 imaging days, dedicated brain PET was performed at 2 time points, 1 and 3 h after 18 F-FLT administration. Tumor SUVs and related parameters were measured at a central laboratory using various volumes of interest: isocontour at 30% of the maximum pixel (SUV mean_30% ), gradient-based segmentation (SUV mean_gradient ), the maximum pixel (SUV max ), and a 1-mL sphere at the region of highest uptake (SUV peak ). Repeatability coefficients (RCs) were calculated from the relative differences between corresponding SUV measurements obtained on the 2 d. Results: RCs for tumor SUVs were 22.5% (SUV mean_30% ), 23.8% (SUV mean_gradient ), 23.2% (SUV max ), and 18.5% (SUV peak ) at 1 h after injection. Corresponding data at 3 h were 22.4%, 25.0%, 27.3%, and 23.6%. Normalizing the tumor SUV data with reference to a background region improved repeatability, and the most stable parameter was the tumor-to-background ratio derived using SUV peak (RC, 16.5%). Conclusion: SUV quantification of 18 F-FLT uptake in glioma had an RC in the range of 18%-24% when imaging began 1 h after 18 F-FLT administration. The volume-of-interest methodology had a small but not negligible influence on repeatability, with the best performance obtained using SUV peak . Although changes in 18 F-FLT SUV after treatment cannot be directly interpreted as a change in tumor proliferation, we have established ranges beyond which SUV differences are likely due to legitimate biologic effects.Key Words: repeatability; reproducibility; FLT; PET; SUV; glioma Nucl Med 2017; 58:393-398 DOI: 10.2967/jnumed.116.178434 Ther adiolabeled thymidine analog 39-deoxy-39-18 F-fluorothymidine ( 18 F-FLT) is incorporated into cells and phosphorylated by thymidine kinase 1. Its retention within cells reflects, in part, thymidine kinase activity and is often positively correlated with cellular proliferation. 18 F-FLT PET can allow noninvasive assessment of tumor proliferation (1) and has been used for monitoring response to treatment in various malignancies (2-5). The tracer would seem to be well suited to the study of glioma as it is associated with low uptake in normal brain, frequently leading to high tumor-to-background (T-to-B) contrast compared with the glucose analog 18 F-FDG. However, the use of 18 F-FLT for response assessment in gliomas is complicated because its uptake can be influenced by multiple m...