The primary aim of this study was to use serial ¹⁸F-3′-deoxy-3′-fluorothymidine (FLT) PET/CT to measure tumor cell proliferation during radiotherapy of squamous cell carcinoma (SCC) of the esophagus. Twenty-one patients with inoperable locally advanced SCC of the esophagus underwent serial ¹⁸F-FLT PET/CT during radiotherapy. Each patient received a pretreatment scan, followed by 1–3 scans after delivery of 2, 6, 10, 20, 30, 40, 50, or 60 Gy to the tumor. Among the 19 patients who completed radiotherapy without interruption, parameters reflecting ¹⁸F-FLT uptake in the tumor (i.e., maximum tumor standardized uptake value [SUVmax] and proliferation target volume) decreased steadily. All patients demonstrated an almost complete absence of proliferating esophageal tumor after 30 Gy and a complete absence after 40 Gy. In the 2 patients whose radiotherapy course was interrupted, ¹⁸F-FLT uptake in the tumor was greater after the interruption than before the interruption. Marked early reduction of ¹⁸F-FLT uptake in irradiated bone marrow was observed in all patients, even after only 2 Gy. All showed a complete absence of proliferating marrow in irradiated regions after 10 Gy. Both patients who underwent scans after completing the entire radiotherapy course showed no tumor uptake on ¹⁸F-FLT PET/CT but high uptake on ¹⁸F-FDG PET/CT. Pathologic examination of these regions revealed inflammatory infiltrates but no residual tumor. ¹⁸F-FLT uptake can be used to monitor the biologic response of esophageal SCC and normal tissue to radiotherapy. Increased uptake of ¹⁸F-FLT after treatment interruptions may reflect accelerated repopulation. ¹⁸F-FLT PET/CT may have an advantage over ¹⁸F-FDG PET/CT in differentiating inflammation from tumor.