Notch signaling plays a critical role in a variety of developmental programs. In vertebrates, the complexity of the process is underscored by the existence of multiple Notch receptors and multiple ligands, each of which displays a distinct expression profile. Furthermore, the ligands can be subdivided into two families, the Serrate/Jagged family and the Delta family. Here we present the isolation of a novel Notch ligand, Delta4. Expression analyses indicate that mouse Delta4 is highly expressed in the eye and lung during embryogenesis and in the heart, lung, liver, and kidney of the adult. Functionally, Delta4 is indistinguishable from Jagged1 in its abilities to inhibit myogenesis and to stimulate transcription through Notch1 and the DNA binding protein CSL.