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 alpha2 (GFR alpha2), RET, and/or NCAM (neural cell adhesion molecule). GFR alpha2 is alternatively spliced into at least three isoforms (GFR alpha2a, GFR alpha2b, and GFR alpha2c). 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 alpha2a and GFR alpha2c, but not GFR alpha2b, promoted neurite outgrowth in transfected Neuro2A cells. These isoforms showed ligand selectivity in MAPK (mitogen-activated protein kinase) [ERK1/2 (extracellular signal-regulated kinase 1/2)] and Akt signaling. In addition, the GFR alpha2 isoforms regulated different early-response genes when stimulated with GDNF or NTN. In coexpression studies, GFR alpha2b was found to inhibit ligand-induced neurite outgrowth by GFR alpha2a and GFR alpha2c. Stimulation of GFR alpha2b also inhibited the neurite outgrowth induced by GFR alpha1a, another member of the GFR alpha. Furthermore, activation of GFR alpha2b 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 alpha2 receptor spliced isoforms, GDNF and NTN may promote or inhibit neurite outgrowth through the multicomponent receptor complex.