The RET receptor tyrosine kinase controls kidney organogenesis and development of subpopulations of enteric and sensory neurons in different vertebrate species, including humans, rodents, chicken and zebrafish. RET is activated by binding to a ligand complex formed by a member of the glial cell line-derived neurotrophic factor (GDNF) family of neurotrophic factors bound to its cognate GFRalpha GPI-linked co-receptor. Despite the absence of GDNF or GFRalpha molecules in the Drosophila genome, a RET orthologue (dRET) has recently been described in this organism and shown to be expressed in subpopulations of cells of the excretory, digestive and nervous systems, thus resembling the expression pattern of RET in vertebrates. In this study, we report on the initial biochemical and functional characterization of the dRET protein in cell culture systems. Full-length dRET could be produced in mammalian and insect cells. Similar to its human counterpart (hRET), overexpression of dRET resulted in its ligand-independent tyrosine phosphorylation, indicating that it bears an active tyrosine kinase. Unlike hRET, however, the extracellular domain of dRET was unable to interact with mammalian GDNF and GFRalpha1. Self association between dRET molecules could neither be detected, indicating that dRET is incapable of mediating cell adhesion by homophilic interactions. A chimeric molecule comprising the extracellular domain of hRET and the kinase domain of dRET was constructed and used to probe ligand-mediated downstream activities of the dRET kinase in PC12 cells. GDNF stimulation of cells transfected with the hRET/dRET chimera resulted in neurite outgrowth comparable to that obtained after transfection of wild-type hRET. These results indicate significant conservation between the biological effects elicited by the human and Drosophila RET kinases, and suggest functions for dRET in neuronal differentiation in the fly.