The molecular mechanism underlying epidermal growth factor receptor (EGFR) localization in mitochondria remains largely unknown. Using immune electron microscopy, we validated that EGFR could be localized on either the outer or the inner membrane of mitochondria. Mutant receptor lacked amino acids 646-660 was flawed in migration onto the organelles, whereas the mutated receptor with a defective endocytosis showed a greater capability of moving onto mitochondria upon stimulation of epidermal growth factor (EGF). Gefitinib, an inhibitor of EGFR kinase, inhibited the receptor endocytosis after short time of treatment, yet, only reduced cell viability as well as the amount of mitochondrial EGFR after longer time of exposure. Moreover, the content of mitochondrial EGFR transfer was decreased when the cells were exposed to the apoptotic inducer etoposide. EGF-induced programmed cell death usually coincided with a decline in mitochondrial EGFR. These data indicated that the mitochondrial-localized EGFR is independent of its internalization and may be correlated with cell survival and participate in the ligand-induced programmed cell death.