Recent studies show that neuronal and glial plasticity are important for the therapeutic action of antidepressants. Here, we demonstrated that amitriptyline, a tricyclic antidepressant, significantly increased GDNF mRNA and GDNF release in C6 cells. Furthermore, different classes of antidepressants increased GDNF release, but non-antidepressant psychotropic drugs did not. The amitriptyline-induced GDNF release was completely inhibited by U0126, a mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor, but was not inhibited by H-89, a protein kinase A inhibitor or calphostin C, a protein kinase C inhibitor. These results suggest that the amitriptyline-induced GDNF release may be regulated through a MEK/MAPK pathway. Next, we examined the effects of monoamines on GDNF release, because antidepressants are known to increase monoamines. 5-HT increased GDNF mRNA and GDNF release, but noradrenaline and dopamine did not. The 5-HT-induced GDNF release was partially, but significantly, blocked by ketanserin, a 5-HT2A receptor antagonist. The 5-HT-induced GDNF release was completely inhibited by U0126, but was not inhibited by H-89 or calphostin C. These results suggest that the 5-HT-induced GDNF release was mediated through a MEK/MAPK pathway and, at least, 5-HT2A receptors. GDNF, as well as other neurotrophic factors, may contribute to explain the therapeutic action of antidepressants and suggest a novel strategy of pharmacological intervention.