The Eph receptors are the largest known family of receptor tyrosine kinases and are notable for distinctive expression patterns in the nervous system and in early vertebrate development. However, all were identified as orphan receptors, and only recently have there been descriptions of a corresponding family of ligands. We describe here a new member of the Eph ligand family, designated ELF-2 (Eph ligand family 2). The cDNA sequence for mouse ELF-2 indicates that it is a transmembrane ligand. It shows closest homology to the other known transmembrane ligand in the family, ELK-L/LERK-2/Cek5-L, with 57% identity in the extracellular domain. There is also striking homology in the cytoplasmic domain, including complete identity of the last 33 amino acids, suggesting intracellular interactions. On cell surfaces, and in a cell-free system, ELF-2 binds to three closely related Eph family receptors, Elk, Cek10 (apparent ortholog of Sek-4 and HEK2), and Cek5 (apparent ortholog of Nuk/Sek-3), all with dissociation constants of approximately 1 nM. In situ hybridization of mouse embryos shows ELF-2 RNA expression in a segmental pattern in the hindbrain region and the segmenting mesoderm. Comparable patterns have been described for Eph family receptors, including Sek-4 and Nuk/Sek-3, suggesting roles for ELF-2 in patterning these regions of the embryo.