DSL proteins are transmembrane ligands of the Notch receptor. They associate with a RING (really interesting new gene) family E3 ubiquitin ligase, either Neuralized (Neur) or Mindbomb 1 (Mib1), as a prerequisite to signaling. Although Neur and Mib1 stimulate internalization of DSL ligands, it is not known how ubiquitylation contributes to signaling. We present a molecular dissection of the intracellular domain (ICD) of Drosophila melanogaster Delta (Dl), a prototype DSL protein. Using a cell-based assay, we detected ubiquitylation of Dl by both Neur and Mib1. The two enzymes use distinct docking sites and displayed different acceptor lysine preferences on the Dl ICD. We generated Dl variants that selectively perturb its interactions with Neur or Mib1 and analyzed their signaling activity in two in vivo contexts. We found an excellent correlation between the ability to undergo ubiquitylation and signaling. Therefore, ubiquitylation of the DSL ICD seems to be a necessary step in the activation of Notch.