The receptor tyrosine kinase Ret plays a critical role in the development of the mammalian excretory and enteric nervous systems. Differential splicing of the primary Ret transcript results in the generation of two main isoforms, Ret9 and Ret51, whose C-terminal amino acid tails diverge after tyrosine (Y) 1062. Monoisoformic mice expressing only Ret9 develop normally and are healthy and fertile. In contrast, animals expressing only Ret51 have aganglionosis of the distal gut and hypoplastic kidneys. By generating monoisoformic mice in which Y1062 of Ret9 has been mutated to phenylalanine, we demonstrate that this amino acid has a critical role in Ret9 signaling that is necessary for the development of the kidneys and the enteric nervous system. These findings argue that the distinct activities of Ret9 and Ret51 result from the differential regulation of Y1062 by C-terminal flanking sequences. However, a mutation which places Y1062 of Ret51 in a Ret9 context improves only marginally the ability of Ret51 to support renal and enteric nervous system development. Finally, monoisoformic mice expressing a variant of Ret9 in which a C-terminal PDZ-binding motif was mutated develop normally and are healthy. Our studies identify Y1062 as a critical regulator of Ret9 signaling and suggest that Ret51-specific motifs are likely to inhibit the activity of this isoform.