Glial cell line-derived neurotrophic factor (GDNF) promotes the survival or differentiation of several types of neurons. This study examines GDNF-induced signal transduction and biological effects in cultured striatal neurons. Results show that GDNF addition to striatal cultures transiently increased the protein levels of phosphorylated p42/p44, but did not change the levels of phosphorylated Akt. GDNF effects on phosphorylated p42/p44 levels were blocked by the mitogen-activated protein kinase (MAPK) pathway specific inhibitors (PD98059 and U0126). Activation of the p42/p44 MAPK pathway by GDNF led to an increase in the degree of dendritic arborization and axon length of both GABA- and calbindin-positive neurons but had no effect on their survival and maturation. These GDNF-mediated effects were suppressed in the presence of the inhibitor of the MAPK pathway (PD98059). Furthermore, the addition of the phosphatidylinositol 3-kinase pathway specific inhibitor (LY294002) blocked GDNF-mediated striatal cell differentiation suggesting that the basal activity of this pathway is needed for the effects of GDNF. Our results indicate that treatment of cultured striatal cells with GDNF specifically activates the p42/p44 MAPK pathway, leading to an increase in the arborization of GABA- and calbindin-positive neurons. © 2005 Wiley-Liss, Inc.