AbstractFibroblast growth factors (FGF) are multifunctional, heparin binding polypeptides that share structural similarity, but differ in their target cell specificity and expression pattern. Here we describe the cloning and expression of the mouse homologue of FGF9, and the use of a panel of soluble FGF receptors and genetically engineered cells to study its receptor binding specificity. FGF9 is found to bind with high affinity (kd: 0.25 nM) to FGFR3, for which a specific ligand has not yet been identified. FGF9 can also bind, albeit with a lower affinity, to FGFR2 but does not bind FGFR1 or FGFR4. There is no significant binding to either FGFR3 or FGFR2, expressed either as soluble receptors or in heparan sulfate deficient cells, in the absence of heparin. Moreover, receptor binding of FGF9 requires heparin in a manner specific to the receptor type. In conclusion FGF9 presents a unique case of ligand-receptor specificity and fulfills the criteria as a high affinity, heparin-dependent ligand for FGFR3.