In the present study, we examined EGF-induced internalization, degradation and trafficking of the epidermal growth factor receptor (EGFR) mutated at serines 1046, 1047, 1057 and 1142 located in its cytoplasmic carboxy-terminal region. We found the serine-mutated EGFR to be inhibited in EGF-induced internalization and degradation in NIH3T3 cells. We therefore tested the hypothesis that these mutations affect ligand-induced c-Cbl association with the receptor, leading to inhibited receptor ubiquitination. EGF was unable to induce ubiquitination of the serine-mutated EGFR, yet EGF-induced phosphorylation of the c-Cbl-binding site at tyrosine 1045, and c-Cbl-EGFR association, was unaffected. To compare the relevance of these serine residues with tyrosine 1045 in their regulation of c-Cbl binding and receptor ubiquitination, we analysed an EGFR mutated at tyrosine 1045 (Y1045F). EGF-induced c-Cbl-EGFR binding was partially inhibited, and receptor ubiquitination was abrogated in cells expressing Y1045F-EGFR. In contrast, ligand-induced internalization and degradation of the Y1045F mutant was similar to that of wild-type EGFR. Together, our data indicate that the serine residues and tyrosine 1045 are essential for EGF-induced receptor ubiquitination, but only the serine residues are critical for EGFR internalization and degradation.