In this study the preparation of detergent resistant membranes (DRMs) and the immunoisolation of intracellular vesicles enriched in rafts marker were used to investigate the effect of physiological doses of EGF in vivo on the compartmentalization and activation of EGFR in rat liver endosomes. Both of these techniques show that following 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 to 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.