We have investigated whether reverse signaling via a glycosyl-phosphatidylinositol (GPI)-linked ephrin controls the behavior of migratory neurons in vivo. During the formation of the enteric nervous system (ENS) in the moth Manduca, approximately 300 neurons [enteric plexus (EP) cells] migrate onto the midgut via bilaterally paired muscle bands but avoid adjacent midline regions. As they migrate, the EP cells express a single ephrin ligand (MsEphrin; a GPI-linked ligand), whereas the midline cells express the corresponding Eph receptor (MsEph). Blocking endogenous MsEphrin-MsEph receptor interactions in cultured embryos resulted in aberrant midline crossing by the neurons and their processes. In contrast, activating endogenous MsEphrin on the EP cells with dimeric MsEph-Fc constructs inhibited their migration and outgrowth, supporting a role for MsEphrin-dependent reverse signaling in this system. In short-term cultures, blocking endogenous MsEph receptors allowed filopodia from the growth cones of the neurons to invade the midline, whereas activating neuronal MsEphrin led to filopodial retraction. MsEphrin-dependent signaling may therefore guide the migratory enteric neurons by restricting the orientation of their leading processes. Knocking down MsEphrin expression in the EP cells with morpholino antisense oligonucleotides also induced aberrant midline crossing, consistent with the effects of blocking endogenous MsEphrin-MsEph interactions. Unexpectedly, this treatment enhanced the overall extent of migration, indicating that MsEphrin-dependent signaling may also modulate the general motility of the EP cells. These results demonstrate that MsEphrin-MsEph receptor interactions normally prevent midline crossing by migratory neurons within the developing ENS, an effect that is most likely mediated by reverse signaling through this GPI-linked ephrin ligand.