Fringe proteins are beta1,3-N-acetylglucosaminyltransferases that modulate signaling through Notch receptors by modifying O-linked fucose on epidermal growth factor domains. Fringe is highly conserved, and comparison among 18 different Fringe proteins from 11 different species identifies a core set of 84 amino acids that are identical among all Fringes. Fringe is only distantly related to other glycosyltransferases, but analysis of the predicted Drosophila proteome identifies a set of four sequence motifs shared among Fringe and other putative beta1,3-glycosyltransferases. To gain functional insight into these conserved sequences, we genetically and molecularly characterized 14 point mutations in Drosophila fringe. Most nonsense mutations act as recessive antimorphs, raising the possibility that Fringe may function as a dimer. Missense mutations identify two distinct motifs that are conserved among beta1,3-glycosyltransferases, and that can be modeled onto key motifs in the crystallographic structures of bovine beta1,4-galactosyltransferase 1 and human glucuronyltransferase I. Other missense mutations map to amino acids that are conserved among Fringe proteins, but not among other glycosyltransferases, and thus may identify structural motifs that are required for unique aspects of Fringe activity.