Patients suspected of recurrent differentiated thyroid cancer (DTC) may require "blind" 131I therapy, with the disadvantage of unpredictable efficacy and toxicity. Alternatively, positron emission tomography (PET) with [18F]fluorodeoxyglucose (18FDG) can document the recurrence, thereby rationalizing therapeutic options. This study compared 18FDG uptake in vivo with biomarkers expected to be involved in the underlying biological mechanisms. Additionally, we investigated whether such features were present in the primary tumors. Preoperatively, 19 patients with recurrent DTC underwent PET. 18FDG uptake was compared with histological and immunohistochemical features in surgical specimens of recurrent and primary tumor. Thirteen of 19 recurrences were positive at PET, and 18FDG uptake was associated with the expression of hexokinase type I (HK I; P = 0.011). All lesions with HK I overexpression were positive on 18FDG PET. HK I expression in the original primary tumor and the metastases was similar in 82% (p = 0.648; P = 0.005). In suspected recurrent thyroid cancer, stratification for 18FDG PET may benefit from pretest immunohistochemical analysis of HK I of the primary tumor, as HK I negativity indicates a low likelihood of PET positivity.