The epidermal growth factor receptor (EGFR) harbors a calmodulin (CaM)‐binding domain (CaM‐BD) and a CaM‐like domain (CaM‐LD) upstream and downstream, respectively, of the tyrosine kinase (TK) domain. We demonstrate in this paper that deletion of the positively charged CaM‐BD (EGFR/CaM‐BD∆) inactivated the TK activity of the receptor. Moreover, deletion of the negatively charged CaM‐LD (EGFR/CaM‐LD∆), leaving a single negative residue (glutamate), reduced the activity of the receptor. In contrast, substituting the CaM‐LD with a histidine/valine‐rich peptide (EGFR/InvCaM‐LD) caused full inactivation. We also demonstrated using confocal microscopy and flow cytometry that the chimera EGFR‐green fluorescent protein (GFP)/CaM‐BD∆, the EGFR/CaM‐LD∆, and EGFR/InvCaM‐LD mutants all bind tetramethylrhodamine‐labelled EGF. These EGFR mutants were localized at the plasma membrane as the wild‐type receptor does. However, only the EGFR/CaM‐LD∆ and EGFR/InvCaM‐LD mutants appear to undergo ligand‐dependent internalization, while the EGFR‐GFP/CaM‐BD∆ mutant seems to be deficient in this regard. The obtained results and in silico modelling studies of the asymmetric structure of the EGFR kinase dimer support a role of a CaM‐BD/CaM‐LD electrostatic interaction in the allosteric activation of the EGFR TK. [ABSTRACT FROM AUTHOR]