Epidermal growth factor receptor (EGFR) plays an important role in cell division and cancer progression, as well as angiogenesis and metastasis. Since many tumor cells exhibit the EGFR on their surface, functional imaging of EGFR provides not only a non-invasive, reproducible, quantifiable alternative to biopsies, but it also greatly complements pharmacokinetic studies by correlating clinical responses with biological effects. Moreover, molecular endpoints of anti-EGFR therapy could be assessed effectively. C225 is a chimeric monoclonal antibody that targets the human extracellular EGFR and inhibits the growth of EGFR-expressing tumor cells. Also, it has been demonstrated that C225, in combination with chemotherapeutic drugs or radiotherapy, is effective in eradicating well-established tumors in nude mice. We have developed99mTc-labeled C225 using ethylenedicysteine (EC) as a chelator. This study aimed at measuring uptake of99mTc–EC–C225 in EGFR+tumor-bearing animal models and preliminary feasibility of imaging patients with head and neck carcinomas.In vitroWestern blot analysis and cytotoxicity assays were used to examine the integrity of EC–C225. Tissue distribution studies of99mTc–EC–C225 were evaluated in tumor-bearing rodents at 0.5–4 h.In vivobiodistribution of99mTc–EC–C225 in tumor-bearing rodents showed increased tumor-to-tissue ratios as a function of time.In vitroand biodistribution studies demonstrated the possibility of using99mTc–EC–C225 to assess EGFR expression. SPECT images confirmed that the tumors could be visualized with99mTc–EC–C225 from 0.5 to 4 h in tumor bearing rodents. We conclude that99mTc–EC–C225 may be useful to assess tumor EGFR expression. This may be useful in the future for selecting patients for treatment with C225.