Human epidermal growth factor receptor (EGFR) plays an important role in homeostatic proliferation of epithelial cells. Mutations and gene amplification may result in overexpression of EGFR, and consecutively increased cell growth and proliferation. Hence, EGFR is associated with various types of cancer. Epidermal growth factor (EGF) and EGF-like ligands bind to EGFR inducing receptor oligomerization and activation. The mechanism of activation in the cell membrane is well understood, and monoclonal antibody therapies targeting membrane-localized EGFR to block binding of EGF are widely used. Based on our fluorescence anisotropy results (measured on transfected cell line), we suggest that cells expressing both EGFR and EGF, the receptor activation may occur intracellularly in the Golgi apparatus. In such case, the membrane-bound EGFR-targeting drugs may not be sufficient in cancer therapy, and drugs targeting intracellularly activated receptors may be required.