Robo3 is a member of the roundabout (Robo) family of proteins that plays a key role in axon guidance and cell migration in the developing nervous system. Recent studies have shown that Robo3 plays a crucial role in controlling axon guidance at the midline of the CNS. Here we describe and compare two human Robo3 isoforms, Robo3A and Robo3B, which differ by the insertion of 26 amino acids at the N-terminus, and these forms appear to be evolutionary conserved. We investigated the bioactivity of these isoforms and show that they have different binding properties to Slit, and that orthologs of these forms are expressed in the mouse embryo. In addition, we show that, like other members of the Robo family, Robo3 can bind homophilically, but it is also capable of binding heterophilically to Robo1 and NCAM. We propose that these properties of Robo3 may contribute to its function at the midline of the CNS.