The epidermal growth factor (EGF)-induced removal of the EGF receptor (EGFR) from the plasma membrane and its endocytic downregulation is a major negative feedback mechanism controlling the intensity and duration of receptor signaling. Different mechanisms of ligand-accelerated endocytosis, rapid ubiquitination of activated EGFR, and sorting of the receptor into multivesicular bodies for lysosomal degradation, are the underlying principles of EGFR downregulation. The physical degradation of the EGFR is thought to protect cells from excessive stimulation. In addition, sequestering the receptor into intralumenal vesicles of endosomes, thereby uncoupling the intracellular tyrosine kinase domain from its downstream effectors, is proposed to contribute to signal attenuation. On the other hand, endosomal EGFR can be active and is able to compensate for signaling initiated at the plasma membrane. How the pool of active endosomal receptor is regulated, and to what extend it contributes to the biological response, has not been investigated conclusively to date. In this study, we aimed to dissect the precise contribution of endocytic sorting events to the EGF response. Consequences of perturbations in EGFR sorting, particularly upon interfering with clathrin- and dynamin-dependent endocytosis, after knockdown of CBL ubiquitin ligases, and of depletion of endosomal sorting complex required for transport (ESCRT) subunits, were investigated in detail. The activation status of signaling components was determined, and a reporter assay was set up to measure EGF-dependent transcriptional activation in living cells. The induction of endogenous target genes downstream of the EGFR-MAPK (mitogen-activated protein kinase) cascade was quantified by real-time RT-PCR, microarray analysis, and by utilizing the recently developed NanoString technology.