The c-ret protooncogene, RET, encodes a receptor tyrosine kinase. RET is activated by members of the glial cell line-derived neurotrophic factor (GDNF) family of ligands, which include GDNF, neurturin, artemin, and persephin. The ligands bind RET through GDNF family receptor α, termed GFRα1–4. Despite the importance of RET signaling in the development of the enteric nervous system and the kidney, the differential signaling mechanisms between RET ligands are poorly established. It has been suggested that signal specificity is achieved through binding of the ligand to its preferred GFRα. To compare the signaling profiles of GDNF and neurturin, we have identified a cell line, NG108-15, which endogenously expresses RET and GFRα1 but not GFRα2–4. Immunoblot data showed that GDNF caused a transient activation, whereas neurturin caused a sustained activation, of both p44/p42 MAP kinases and PLCγ. Under serum starvation, NG108-15 cells differentiate and form neurites. Neurturin but not GDNF stimulated neurite outgrowth, which could be blocked by the selective PLC inhibitor U73122. On the other hand, GDNF but not neurturin promoted cell survival, and this could be blocked by the p44/p42 MAP kinase inhibitor PD98059. Our findings not only show the differential signaling of GDNF and neurturin but also suggest that this can be achieved through binding to the same GFRα subtype, leading to distinct biological responses. © 2005 Wiley-Liss, Inc.