Alternative splicing of signaling proteins can contribute to the complexity of signaling networks. We find that expression of mouse RON, but not human RON, results in constitutive receptor autophosphorylation, ligand-independent activation of the mitogen-activated protein kinase pathway, and association of the receptor with c-Src. Using chimeric receptors, we mapped the region for this difference in signaling capacity of mouse and human RON to the juxtamembrane domain. Expression of these receptors in primary erythroid progenitor cells also demonstrated a functional difference in the ability of mouse and human RON to support erythropoietin-independent colony formation that mapped to the juxtamembrane domain. Splicing of the mouse RON receptor tyrosine kinase transcript results in the constitutive deletion of an exon used by all other known RON orthologs that encodes part of the juxtamembrane domain of the receptor. Mutational analysis indicated that the two tyrosines present in this region in human RON, one of which has been previously shown to be a c-Cbl binding site, are not responsible for this difference. However, deletion of this region in the context of human RON enhanced receptor phosphorylation, activation of mitogen-activated protein kinase, and association of c-Src at levels comparable with those observed with mouse RON. These data provide direct evidence that the divergence of exon usage among different species can generate a protein with novel activity and subsequently add to the complexity of cellular signaling regulation.