Axons of GnRH neurons terminate at the median eminence in the medial basal hypothalamus (MBH) of the brain early in development. Similarly, GnRH neurons in grafts of preoptic area (POA) tissue within the third ventricle of hypogonadal mice preferentially innervate the median eminence. Organotypic cocultures of POA explants with other neural tissues suggest that a soluble substance(s) derived from the MBH may be directing this targeting. To begin to identify diffusable chemoattractants, we used preincubated heparin-coated acrylic beads to present specific solutes to POA explants on collagen- and laminin-coated membranes in insert chambers. GnRH axons grew on the membrane in greater number and with longer axons toward conditioned medium from MBH cultures than on the side away from the beads (P < 0.01). In contrast, GnRH axons showed no preferential outgrowth when incubated with beads soaked in control, defined medium. The attraction of MBH-conditioned medium was not generalizable to all neuroendocrine neurons, as it was not seen for galanin immunoreactive outgrowth from POA explants. There also were more GnRH axons toward conditioned medium from mouse brain microvascular endothelial cells, but no difference in axon length. Basic fibroblast growth factor (bFGF), a component of both endothelial cells and ventricular tanycytes, significantly attracted more and longer GnRH axons. Thus, bFGF may be one of the soluble factors directing GnRH outgrowth to the median eminence. However, as with so many other redundancies in the reproductive system, it is unlikely that it is the only targeting factor, as bFGF knockout mice are reported to be reproductively competent.