The Notch receptor, the extracellular domain of which is composed of epidermal growth factor (EGF) repeats, is activated by Delta and Serrate. How Notch discriminates between these ligands is poorly understood. This study identifies an amino acid in Notch EGF repeat 8 that is essential for the binding of Serrate but not Delta. Val to Met mutation at residue 361 in Drosophila melanogaster EGF repeat 8 (Notchjigsaw) caused similar phenotypes to loss of Serrate function. Further analysis revealed that Notchjigsaw is defective in Serrate−Notch signalling, but not in Delta−Notch signalling, and that this mutation reduces Serrate–Notch binding. Val to Met mutation at residue 327 in mouse Notch 2 decreased its interaction with the Serrate homologue Jagged 1 and also Jagged 1−Notch 2 signalling, whereas Delta-like 1−Notch 2 signalling was unaffected. Thus, the specificity of this Val residue is conserved in Serrate−Notch signalling.