In this study, the preparation of detergent-resistant membranes (DRMs) and the immunoisolation of intracellular vesicles enriched in raft markers were used to investigate the effect of physiological doses of epidermal growth factor (EGF) in vivo on the compartmentalization and activation of EGF receptor (EGFR) in rat liver endosomes. Both of these techniques show that after EGF administration, a distinctive population of intracellular EGFR, which was characterized by a high level of tyrosine phosphorylation, accumulated in endosomes. EGFR recruited to early endosomes were more tyrosine phosphorylated than those from late endosomes. However, the level of tyrosine phosphorylation of EGFR in DRMs isolated from early and late endosomes was comparable, suggesting that EGFR in endosomal DRMs are more resistant to tyrosine dephosphorylation. In accordance with the higher level of Tyr phosphorylation, EGF induced an augmented recruitment of Grb2 and Shc to endosomal DRMs compared with whole endosomes. Furthermore, a proteomic analysis identified a selective increase of many alpha-subunits of heterotrimeric G proteins in endosomal DRMs in response to EGF. These observations suggest that a distinctive pool of endocytic EGFR, potentially competent for signaling, is actively trafficking through intracellular compartments with the characteristic of lipid rafts.