Abstract The primary pharmacologic treatment for depression over the past several decades has been drugs that inhibit synaptic reuptake of monoamine neurotransmitters. Although the importance of monoamine neurotransmission in antidepressant efficacy cannot be discounted, other neurotransmitter systems are known to contribute to the mechanism of action of antidepressants. Furthermore, the existence of a large group of non-responding patients necessitates development of novel compounds to treat depression. A growing body of data suggests that cholinergic systems may be potential targets for development of novel antidepressant compounds, and that excessive cholinergic signaling may contribute to the pathophysiology of depression. Depression can be conceptualized as a maladaptive response in which the ensemble of stress-related behaviors does not switch back to the collection of behaviors that allow exploration of the environment and pursuit of natural rewards. Neuromodulators are well placed to coordinate these ensembles of behavior and to mediate the switch between coordinated behavioral states. ACh facilitates stress-related learning and hypervigilance through actions in the amygdala and promotes stress-related avoidance mediated through the hippocampus. The effects in each of these brain areas are distinct and mediated through different receptor subtypes, and we propose that these can be bound into an ensemble of stress-related behaviors by release of ACh. We therefore hypothesize that increasing ACh signaling in amygdala and hippocampus facilitates the transition to this stress-related behavioral ensemble and consequently, blocking cholinergic receptors of different kinds promotes an adaptive, antidepressant response. In these studies we will identify neuronal mechanisms in amygdala underlying cholinergic regulation of stress-related behaviors, test the hypothesis that phasic increases in ACh signaling in hippocampus, as occurs in response to stress, induces behaviors related to depression, and determine whether distinct populations of ACh neurons projecting to amygdala and hippocampus are responsible for the effects of cholinergic signaling on stress-induced behaviors.