Dissociated parasympathetic neurons rapidly initiate neurite outgrowth when exposed to culture medium previously conditioned by the growth of embryonic heart cells. The inducer of neurite outgrowth in the conditioned medium is a substratum-conditioning factor; that is, it does not act in a soluble form, but acts only when bound to the nerve cell culture substratum. When a sharp border is created between a region of the substratum coated with this factor and a region coated with unconditioned medium, neurites fail to cross this border; rather, they change their direction of outgrowth so as to remain on the conditioned substratum. Thus, long after the initiation of outgrowthhas been induced, elongating neurites continue to respond to the substratum-conditioning factor in a manner that allows their outgrowth to be channeled along a pathway of this neurotropic substratum-associated material.