AbstractNeural representations of the environment within the brain take the form of topographic maps whose formation relies on graded expression of axon guidance molecules. Retinocollicular map formation, from retinal ganglion cells (RGCs) to the superior colliculus (SC) in the midbrain, is mainly driven by Eph receptors and their ligands ephrins. However, other guidance molecules participate in the formation of this map. Here we demonstrate that the receptor Neuropilin‐2 is expressed in an increasing nasal–temporal gradient in RGCs, whereas one of its ligands, Semaphorin3F, but not other Sema3 molecules, presents a graded low‐rostral to high‐caudal expression in the SC when mapping is underway. Neuropilin‐2 and its coreceptor Plexin A1 are present on RGC growth cones. Collapse assays demonstrate that Semaphorin3F induces significant growth cone collapse of temporal, but not nasal, RGCs expressing high levels of Neuropilin‐2. Our results suggest that Neuropilin‐2/Semaphorin3F are new candidates involved in retinotopy formation within the SC. Developmental Dynamics 237:3394–3403, 2008. © 2008 Wiley‐Liss, Inc.