ROBO1 is a 170-200 kDa member of the ROBO family of guidance molecules. It serves as a repulsing molecule that prevents axons from crossing the midline in the developing central nervous system (CNS) (1). Mature rat ROBO1 consists of an 872 amino acid (aa) extracellular domain (ECD), a 21 aa transmembrane segment, and a 733 aa cytoplasmic region (2). The ECD contains five tandem Ig-like domains followed by three tandem fibronectin type III domains. Usage of an alternative promoter for human ROBO1 generates a shorter isoform (ROBO1b or DUTT1) that has an 18 aa substitution for the N-terminal 57 residues (2, 3). Within the ECD, rat ROBO1 shares 98% and 97% aa identity with mouse and human ROBO1, respectively. A 120 kDa soluble fragment of the ECD can be shed by juxtamembrane proteolytic cleavage (4). ROBO1 is expressed on pioneer axons in the neural tube and proliferative zones of the cortical plate (5-8). It is restricted to axonal growth cones that do not cross the midline and to midline-crossing axons only after they cross (2). ROBO1b shows even wider expression in the developing mouse CNS and is the predominant isoform in spinal cord commissural axons (5). ROBO1 plays an important role in axon pathfinding and tract development (6, 9, 10) as well as in neuronal proliferation and dendrite branching (7, 8). ROBO1 binds to Slit1 and Slit2, leading to growth cone collapse and axonal branching (7, 11). It also regulates Netrin-1 and Semaphorin 3A and 3F responsiveness through its ability to form in cis complexes with Neuropilin-1, Neuropilin-2, Plexin A1, DCC, and FLRT3 (10, 12, 13). ROBO1 is additionally expressed in developing ductal epithelia where its binding to Slit2 restricts branching morphogenesis (14). ROBO1-Slit2 interactions can also suppress tumorigenesis (15), enhance VEGF-induced angiogenesis (16), and enhance CXCL12 induced T cell chemotaxis by associating with CXCR4 (17).