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Abstract Background Ubiquitylation of the ligands and the receptor plays an important part in the regulation of the activity of the evolutionary conserved Notch signalling pathway. However, its function for activation of Notch is not completely understood, despite the identification of several E3 ligases devoted to the receptor. Results Here we analysed a variant of the Notch receptor where all lysines in its intracellular domain are replaced by arginines. Our analysis of this variant revealed that ubiquitylation of Notch is not essential for its endocytosis. We identified two functions for ubiquitylation of lysines in the Notch receptor. First, it is required for the degradation of free Notch intracellular domain (NICD) in the nucleus, which prevents a prolonged activation of the pathway. More importantly, it is also required for the incorporation of Notch into intraluminal vesicles of maturing endosomes to prevent ligand-independent activation of the pathway from late endosomal compartments. Conclusions The findings clarify the role of lysine-dependent ubiquitylation of the Notch receptor and indicate that Notch is endocytosed by several independent operating mechanisms.