AbstractMany receptor tyrosine kinases (RTKs) can be detected in the cell nucleus, such as EGFR, HER‐2, HER‐3, HER‐4, and fibroblast growth factor receptor. EGFR, HER‐2 and HER‐4 contain transactivational activity and function as transcription co‐factors to activate gene promoters. High EGFR in tumor nuclei correlates with increased tumor proliferation and poor survival in cancer patients. However, the mechanism by which cell‐surface EGFR translocates into the cell nucleus remains largely unknown. Here, we found that EGFR co‐localizes and interacts with importins α1/β1, carriers that are critical for macromolecules nuclear import. EGFR variant mutated at the nuclear localization signal (NLS) is defective in associating with importins and in entering the nuclei indicating that EGFR's NLS is critical for EGFR/importins interaction and EGFR nuclear import. Moreover, disruption of receptor internalization process using chemicals and forced expression of dominant‐negative Dynamin II mutant suppressed nuclear entry of EGFR. Additional evidences suggest an involvement of endosomal sorting machinery in EGFR nuclear translocalization. Finally, we found that nuclear export of EGFR may involve CRM1 exportin as we detected EGFR/CRM1 interaction and markedly increased nuclear EGFR following exposure to leptomycin B, a CRM1 inhibitor. Collectively, these data suggest the importance of receptor endocytosis, endosomal sorting machinery, interaction with importins α1/β1, and exportin CRM1 in EGFR nuclear‐cytoplasmic trafficking. Together, our work sheds light into the nature and regulation of the nuclear EGFR pathway and provides a plausible mechanism by which cells shuttle cell‐surface EGFR and potentially other RTKs through the nuclear pore complex and into the nuclear compartment. J. Cell. Biochem. © 2006 Wiley‐Liss, Inc.