The epidermal growth factor receptor (EGFR) is a member of the erbB family of receptor tyrosine kinases. In our study, we were interested to study the intracrine signaling mechanism of the EGF receptor in cells expressing both EGFR and its ligand EGF and in the Golgi apparatus. An anti-phospho-EGFR antibody was used to identify receptor phosphorylation in the Golgi, which was visualized by a TagBFP-labeled giantin, a Golgi- resident protein. FLIM-FRET measurements were used to test our hypothesis that EGF ligands can bind to the EGFR in the Golgi. Our results prove the activation and oligomerization of the EGFR in the Golgi. If our hypothesis of the intracrine signaling mechanism of EGFR can occur in Golgi, before the receptor reaches the plasma membrane, the antibody therapies against the extracellular ligand binding domain of the EGFR would be inefficient in cancer cells expressing both EGFR and its ligand.