The glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) (GDNF, neurturin, artemin, and persephin) are critical regulators of neurodevelopment and support the survival of midbrain dopaminergic and spinal motor neurons in vitro and in animal disease models making them attractive therapeutic candidates for treatment of neurodegenerative diseases. The GFLs signal through a multicomponent receptor complex comprised of a high affinity binding component (GDNF-family receptor α-component (GFRα1-GFRα4)) and the receptor tyrosine kinase RET. To begin characterization of GFL receptor specificity at the molecular level, we performed comprehensive homologue- scanning mutagenesis of GDNF, the prototypical member of the GFLs. Replacing short segments of GDNF with the homologous segments from persephin (PSPN) (which cannot bind or activate GFRα1 · RET or GFRα2 · RET) identified sites along the second finger of GDNF critical for activating the GFRα1 · RET and GFRα2 · RET receptor complexes. Furthermore, introduction of these regions from GDNF, neurturin, or artemin into PSPN demonstrated that they are sufficient for activating GFRα · RET, but additional determinants are required for interaction with the other GFRαs. This difference in the molecular basis of GFL-GFRα specificity allowed the production of GFRα1 · RET-specific agonists and provides a foundation for understanding of GFL- GFRα · RET signaling at the molecular level.