Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) are two homologeous proteins that have been recognized as potent survival factors for distinct neuronal populations. GDNF and NTN act through a two-component receptor system consisting of the ligand-specific binding subunits GDNF family receptor (GFR)α-1 and GFRα-2 and the common transducing subunit c-Ret. In addition, it has been demonstrated that GDNF can signal through GFRα-1 in the absence of c-Ret. In the present study, we sought to determine whether a similar c-Ret-independent signaling applies for GFRα-2. In addition, we have characterized the ligand specificity of the c-Ret-independent action of GFRαs. To establish an assay system for these studies, several neural cell lines were screened for the presence of GDNF and NTN receptor subunits by RT-PCR and immunoblot analysis. c-Ret expression was detectable only in Neuro2A cells, which did not express GFRα-1 or GFRα-2. The neuronal cell line LS expressed GFRα-2, and the glial cell line Mes42 expressed GFRα-1, whereas the neuronal cell line B104 expressed both GFRα-1 and GFRα-2. Stimulation of B104 and Mes42 cells with GDNF, but not with NTN, for 10 min resulted in CREB phosphorylation. In apparent contrast, neither NTN nor GDNF promoted CREB activation in LS and Neuro2A cells. Moreover, exposure of LS cells to NTN or GDNF also failed to activate AKT and ERK. Together these findings provide evidence that, in contrast to GFRα-1, GFRα-2 fails to signal in the absence of c-Ret. In addition, these observations reveal that c-Ret-independent signaling of GFRα-1 is ligand- specific and occurs only with GDNF. J. Neurosci. Res. 66:390–395, 2001. © 2001 Wiley-Liss, Inc.