Accumulating evidence indicates that neural activity in the lateral septum (LS) influences the pathophysiology of depression and therapeutic effectiveness of antidepressant drugs. For example, the development of behavioral deficits in animal screens for antidepressant drug activity corresponds with a blunting of LS activity, whereas chronic treatment with antidepressants enhances cell firing in the LS; however, the molecular mechanisms underlying such behavioral functions of the LS have not been determined. The nonreceptor tyrosine kinase Pyk2 is highly expressed in the LS and plays important roles in regulating cellular excitability and synaptic plasticity, making it an attractive candidate for regulating the effects of stress and antidepressants on LS functioning and behavior. We provide evidence that stress decreases Pyk2 phosphorylation in the LS, whereas enhancing Pyk2 expression in LS neurons has an antidepressant effect behaviorally.Pyk2 messenger ribonucleic acid (mRNA) expression in the rat forebrain was detected by in situ hybridization, and a brief description of the distribution of Pyk2 mRNA in selected areas is presented. Levels of total Pyk2 protein and phosphorylated Pyk2 were subsequently measured in the LS and hippocampus following stress exposure, as were levels of extracellular stimuli-regulated kinase (Erk) and phospho-Erk. Herpes simplex virus (HSV)-mediated gene transfer was then used to enhance Pyk2 expression in the LS, and the effect this had on behavior in the learned helplessness model of depression was evaluated. High levels of Pyk2 mRNA were detected in a number of forebrain regions, including the hippocampus and LS. Following acute stress exposure, subjects showed a decrease in phosphorylated Pyk2 and Erk in the LS but not in the hippocampus. Total levels of Pyk2 and Erk remained unchanged following stress. In the learned helplessness paradigm, injection of HSV-Pyk2 into the LS prevented the active avoidance deficit caused by exposure to inescapable shock, indicative of an antidepressant effect. These results indicate that following acute stress, Pyk2 and Erk activity in the LS are decreased, whereas experimentally increasing Pyk2 activity in LS neurons reverses the behavioral deficits of acute, inescapable stress. These findings establish a role for the tyrosine kinase Pyk2 in the biochemical and behavioral responses to stress and suggest a possible role in the pathophysiology of depression, particularly notable considering Pyk2's role in promoting synaptic plasticity.