Brain-derived neurotrophic factor (BDNF) has been strongly implicated in the synaptic plasticity, neuronal survival and pathophysiology of depression. Lithium and valproic acid (VPA) are two primary mood-stabilizing drugs used to treat bipolar disorder. Treatment of cultured rat cortical neurons with therapeutic concentrations of LiCl or VPA selectively increased the levels of exon IV (formerly rat exon III)-containing BDNF mRNA, and the activity of BDNF promoter IV. Surprisingly, lithium- or VPA-responsive element(s) in promoter IV resides in a region upstream from the calcium-responsive elements (CaREs) responsible for depolarization-induced BDNF induction. Moreover, activation of BDNF promoter IV by lithium or VPA occurred in cortical neurons depolarized with KCl, and deletion of these three CaREs did not abolish lithium- or VPA-induced activation. Lithium and VPA are direct inhibitors of glycogen synthase kinase-3 (GSK-3) and histone deacetylase (HDAC), respectively. We showed that lithium-induced activation of promoter IV was mimicked by pharmacological inhibition of GSK-3 or short interfering RNA (siRNA)-mediated gene silencing of GSK-3alpha or GSK-3beta isoforms. Furthermore, treatment with other HDAC inhibitors, sodium butyrate and trichostatin A, or transfection with an HDAC1-specific siRNA also activated BDNF promoter IV. Our study demonstrates for the first time that GSK-3 and HDAC are respective initial targets for lithium and VPA to activate BDNF promoter IV, and that this BDNF induction involves a novel responsive region in promoter IV of the BDNF gene. Our results have strong implications for the therapeutic actions of these two mood stabilizers.