Recently, the changes of neuronal and glial plasticity related gene expression following the increase of monoamine are suggested to be important for the therapeutic effect of antidepressants. We previously showed that antidepressants increased glial cell line-derived neurotrophic factor (GDNF) expression, which was dependent on acute activation of protein tyrosine kinase (PTK) and extracellular signal-regulated kinase (ERK) in rat C6 glioma cells (C6 cells) and normal human astrocytes (NHA). Transcription of many genes including GDNF is directed by the cAMP responsive element (CRE) and its cognate transcription factor CRE binding protein (CREB). In this study, we showed that amitriptyline, a tricyclic antidepressant, acutely increased phosphorylation of CREB, without altering the level of total CREB in C6 cells as well as in NHA. In contrast, acute amitriptyline treatment did not affect phosphorylation of CREB in SH-SY5Y cells, a human neuroblastoma cell line. Different classes of antidepressants as well as amitriptyline acutely increased phosphorylation of CREB, but haloperidol and diazepam did not. The amitriptyline-induced phosphorylation of CREB was completely blocked by U0126 [a mitogen-activated protein (MAP) kinase kinase 1 inhibitor] and genistein (a PTK inhibitor), but not by inhibitors of protein kinase A, p38 MAP kinase, or Ca(2+)/calmodulin-dependent kinase. Amitriptyline treatment also increased the expression of luciferase reporter gene regulated by CRE elements. The amitriptyline-induced luciferase activity was completely inhibited by U0126 in the same as phosphorylation of CREB. These results suggest that antidepressants acutely increase CREB activity in PTK and ERK-dependent manners, which might contribute to gene expression including GDNF in glial cells.