ObjectiveThe nucleus ambiguus (NAmb) is a column of neurons in the medulla oblongata, involved in bulbar functions. Expression of Glial Cell‐Derived Neurotrophic Factor (GDNF) and its receptors (GDNFR) is observed within the cell bodies during reinnervation following recurrent laryngeal nerve (RLN) injury. Little is known regarding GDNFR expression in the formation of the NAmb and the laryngeal innervation during embryogenesis. Understanding the timing and pattern of GDNFR expression in embryogenesis versus after RLN injury may provide insights into therapeutic targets for regeneration after RLN injury.Study DesignLaboratory experiment.MethodsRat brainstems at E14.5/E16.5/E18.5/E20.5/adult were stained for GDNFR: GFRα‐1/GFRα‐2/GFRα‐3/Ret. Islet1 and choline acetyltransferase were used as cell body markers. Sections were observed using fluorescent microscopy and quantified through manual cell counting.ResultsExpression of GFRα‐1, GFRα‐3, and Ret was identified within the NAmb, hypoglossal, and facial nuclei of the adult medulla. During development, GFRα‐1 immunoreactivity was seen at E20.5. GFRα‐2 expression was not observed at any timepoint. GFRα‐3 expression began at E16.5. Ret expression within nerve fibers in the NAmb were observed beginning at E14.5, but never in the cell bodies.ConclusionEmbryonic GDNFR expression in the NAmb differs from that of the adult after RLN injury. The developing brainstem experienced upregulation at discrete timepoints with signaling sustained through adulthood. In contrast, adult RLN‐transected rats experienced patterns of up and down regulation. GFRα‐1 may contribute to muscle targeting and neuromuscular junction maturation, GFRα‐3 may contribute to both, as well as axon guidance. It is likely that GDNF is functioning via a Ret‐independent pathway.Level of EvidenceNA Laryngoscope, 133:2240–2247, 2023