Purpose. Anti-EGFR antibody cetuximab (C225) is used in combination with radiotherapy of head and neck squamous cell carcinoma (HNSCC) patients. We investigated whether conjugation of cetuximab with trans-cyclohexyl-diethylene- triamine-pentaacetic acid (CHX-A''-DTPA) and radiolabeling with ⁹⁰Yttrium affect the molecular and cellular function of cetuximab and improve its combined effect with external-beam irradiation (EBI).Methods. The following cell lines were used: HNSCC UT5, SAS, FaDu, as well as A43, Chinese hamster ovary cells (CHO), and human skin fibroblast HSF7. Binding affinity and kinetics, specificity, retention, and the combination of ⁹⁰Y- cetuximab with EBI were evaluated.Results. Control cetuximab and CHX-A''-DTPA-cetuximab blocked the proliferation activity of UT5 cells. In combination with EBI, CHX-A''-DTPA-cetuximab increased the radiosensitivity of UT5 to a similar degree as control cetuximab did. In contrast, in SAS and HSF7 cells neither proliferation nor radiosensitivity was affected by either of the antibodies. Binding [ ⁹⁰Y]Y-CHX-A''-DTPA-cetuximab ( ⁹⁰Y-cetuximab) to EGFR in HNSCC cells occurred time dependently with a maximum binding at 24 h. Retention of ⁹⁰Y-cetuximab was similar in both HNSCC cell lines; 24 h after treatment, approximately 90% of bound activity remained in the cell layer. Competition assays, using cell membranes in the absence of an internalized fraction of cetuximab, showed that the cetuximab affinity is not lost as a result of conjugation with CHX-A''-DTPA. Cetuximab and CHX-A''-DTPA-cetuximab blocked EGF-induced Y1068 phosphorylation of EGFR. The lack of an effect of cetuximab on EGF-induced Akt and ERK1/2 phosphorylation and the inhibition of irradiation (IR)-induced Akt and ERK1/2 phosphorylation by cetuximab were not affected by DTPA conjugation. ⁹⁰Y-cetuximab in combination with EBI resulted in a pronounced inhibition of colony formation of HNSCC cells.Conclusions. Conjugation of CHX-A''-DTPA to cetuximab does not alter the cellular and biological function of cetuximab. ⁹⁰Y-labeling of cetuximab in combination with EBI may improve radiotherapy outcome.