Roundabout (Robo) receptors and their secreted ligand Slits have been shown to function in a number of developmental events both inside and outside of the nervous system. We previously cloned zebrafish robo orthologs to gain a better understanding of Robo function in vertebrates. Further characterization of one of these orthologs, robo3, has unveiled the presence of two distinct isoforms, robo3 variant 1 (robo3var1) and robo3 variant 2 (robo3var2). These two isoforms differ only in their 5′-ends with robo3var1, but not robo3var2, containing a canonical signal sequence. Despite this difference, both forms accumulate on the cell surface. Both isoforms are contributed maternally and exhibit unique and dynamic gene expression patterns during development. Functional analysis of robo3 isoforms using an antisense gene knockdown strategy suggests that Robo3var1 functions in motor axon pathfinding, whereas Robo3var2 appears to function in dorsoventral cell fate specification. This study reveals a novel function for Robo receptors in specifying ventral cell fates during vertebrate development. © 2005 Elsevier Ireland Ltd. All rights reserved.