Myelin is crucial for the stabilization of the entorhinohippocampal projection during late development and is a non-permissive substrate for regrowing axons after lesion in the adult brain. We used two in vitro assays to analyse the impact of myelin on rat entorhinohippocampal projection neurons. A stripe assay was used to study the impact of myelin on the choice behaviour of axons from the entorhinal cortex (EC). Given a choice between alternating hippocampal membrane lanes from developmental stages ranging from early postnatal to adult, EC axons preferred to extend on early postnatal hippocampal membranes. Neither the neutralization of myelin-associated factors by a specific antibody (IN-1) nor the separation of myelin from membranes interfered with the axons' choice behaviour. The entorhinal axons showed no preference in the membrane combination of adult and myelin-free adult hippocampal membranes. These stripe assay experiments demonstrate that support for EC axon choice in the developing hippocampus is maturation-dependent and is not influenced by myelin. The application of IN-1 in the outgrowth assay and the separation of myelin from membranes, enhanced elongation of outgrowing entorhinal axons on adult hippocampal membranes, whereas a control antibody did not. This shows that myelin-associated factors have a strong inhibitory effect on the outgrowth length of entorhinal axons. In conclusion, we suggest that axonal elongation in the entorhinohippocampal system during development is strongly influenced by myelin-associated growth inhibition factors and that specific target finding of entorhinal axons is regulated by a different mechanism.