Quantitative 3′-deoxy-3′-¹⁸F-fluorothymidine (¹⁸F-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 ¹⁸F-FLT PET as part of a multicenter trial involving patients with high-grade glioma. ¹⁸F-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 ¹⁸F-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. 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%). SUV quantification of ¹⁸F-FLT uptake in glioma had an RC in the range of 18%–24% when imaging began 1 h after ¹⁸F-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 ¹⁸F-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.