Eph-related receptor tyrosine kinases and ligands are expressed at high levels in the developing nervous system, giving rise to the proposal that they are involved in neuronal connection. Cek8 was found to be predominantly expressed on a subset of motoneurons innervating limb but not body muscles during motoneuron axonal growth. Here we show that the ligands RAGS and ELF-1 were expressed in limb buds and that they activated Cek8 when presented in membrane-bound or clustered forms of Fc chimeric proteins but not in unclustered soluble forms. When chick embryonic motoneurons enriched by panning were cultured on clustered forms of RAGS-Fc and ELF-1-Fc, the neutrite growth of motoneurons expressing Cek8 was inhibited. Our results show a relationship between receptor phosphorylation and neurite growth inhibition and suggest that Eph-related kinases and ligands have a regulatory effect on the axon growth of motoneurons during development.