The glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) belong to a structurally related family of neurotrophic factors. NTN exerts its effect through a multicomponent receptor system consisting of the GDNF family receptor ␣2 (GFR␣2), RET, and/or NCAM (neural cell adhesion molecule). GFR␣2 is alternatively spliced into at least three isoforms (GFR␣2a, GFR␣2b, and GFR␣2c). It is currently unknown whether these isoforms share similar functional and biochemical properties. Using highly specific and sensitive quantitative real-time PCR, these isoforms were found to be expressed at comparable levels in various regions of the human brain. When stimulated with GDNF and NTN, both GFR␣2a and GFR␣2c, but not GFR␣2b, promoted neurite outgrowth in transfected Neuro2A cells. These isoforms showed ligand selectivity in MAPK (mitogen-activated protein kinase) [ERK1/2 (extracellular signalregulated kinase 1/2)] and Akt signaling. In addition, the GFR␣2 isoforms regulated different early-response genes when stimulated with GDNF or NTN. In coexpression studies, GFR␣2b was found to inhibit ligand-induced neurite outgrowth by GFR␣2a and GFR␣2c. Stimulation of GFR␣2b also inhibited the neurite outgrowth induced by GFR␣1a, another member of the GFR␣. Furthermore, activation of GFR␣2b inhibited neurite outgrowth induced by retinoic acid and activated RhoA. Together, these data suggest a novel paradigm for the regulation of growth factor signaling and neurite outgrowth via an inhibitory splice variant of the receptor. Thus, depending on the expressions of specific GFR␣2 receptor spliced isoforms, GDNF and NTN may promote or inhibit neurite outgrowth through the multicomponent receptor complex.