Notch signaling is a component of a wide variety of developmental processes in many organisms. Notch activity can be modulated by O-fucosylation (mediated by protein O-fucosyltransferase-1) and Fringe, a ␤1,3-N-acetylglucosaminyltransferase that modifies O-fucose in the context of epidermal growth factor-like (EGF) repeats. Fringe was initially described in Drosophila, and three mammalian homologues have been identified, Manic fringe, Lunatic fringe, and Radical fringe. Here for the first time we have demonstrated that, similar to Manic and Lunatic, Radical fringe is also a fucose-specific ␤1,3-N-acetylglucosaminyltransferase. The fact that three Fringe homologues exist in mammals raises the question of whether and how these enzymes differ. Although Notch contains numerous EGF repeats that are predicted to be modified by O-fucose, previous studies in our laboratory have demonstrated that not all O-fucosylated EGF repeats of Notch are further modified by Fringe, suggesting that the Fringe enzymes can differentiate between them. In this work, we have sought to identify specificity determinants for the recognition of an individual O-fucosylated EGF repeat by the Fringe enzymes. We have also sought to determine differences in the biochemical behavior of the Fringes with regard to their in vitro enzymatic activities. Using both in vivo and in vitro experiments, we have found two amino acids that appear to be important for the recognition of an O-fucosylated EGF repeat by all three mammalian Fringes. These amino acids provide an initial step toward defining sequences that will allow us to predict which O-fucosylated EGF repeats are modified by the Fringes.The Notch protein is a transmembrane receptor involved in various cell fate decisions. It was initially characterized in Drosophila and has subsequently been found in all known metazoans (1). Notch becomes activated upon binding to its ligands located on apposing cells. These ligands, known collectively as the DSL (for Delta-Serrate-Lag2) family, fall into two classes, Serrate/Jagged and Delta. A large portion of the extracellular domain of Notch is composed of epidermal growth factor (EGF) 3 -like repeats, and many of these EGF repeats contain consensus sites for modification by O-fucose (a process mediated by the protein O-fucosyltransferase-1 (O-FucT-1) (2, 3). Some of these O-fucosylated EGF repeats can be further modified by the actions of Fringe, a ␤1,3-GlcNAc transferase (4 -6). The modification of Notch by Fringe plays an important role in the modulation of Notch signaling in various contexts (7,8).Fringe was initially described in Drosophila as a gene that modulates dorsal-ventral cell interactions in the developing wing (9). Further studies reveal that Fringe functions specifically by modulating the response of Notch to its ligands, potentiating Delta signaling while inhibiting Serrate signaling (10 -12). Three mammalian homologues to Drosophila Fringe (Dfng) have been identified, Manic fringe (Mfng), Lunatic fringe (Lfng), and Radical fringe (Rfng). Lfng a...