One of the biggest issues in treating tobacco smoking dependence is the high rate of relapse during early abstinence, which is primarily due to the powerful withdrawal syndrome including cognitive impairment, intense craving, and a poor mood. The neurotransmitters acetylcholine (ACh) and dopamine (DA) are known to mediate the reinforcing effects of smoking and they also drive the primary behavioral withdrawal symptoms; however, the very basic cholinergic and dopaminergic brain mechanisms that underlie withdrawal and relapse in tobacco smokers are unknown. Nicotine, the primary addictive chemical in tobacco smoke, binds to neuronal beta2 subunit-containing nicotinic acetylcholine receptors (?2*-nAChRs) leading to widespread changes in neurotransmitter levels including DA, and an increase in the number of ?2*-nAChRs throughout the brain. We have previously shown that there are dynamic changes in numbers of ?2*-nAChRs over abstinence; but we do not know the functional significance, i.e., if ACh neurotransmission is altered over the course of abstinence, and if altered ACh levels are associated with cognitive dysfunction and relapse vulnerability. A substantial preclinical literature demonstrates that nAChRs and the cholinergic system dynamically control the mesolimbic DA system by enhancing, inhibiting and filtering striatal DA release. A primary issue in tobacco smoking withdrawal is the intense craving in response to cues and we know that DA regulates the salience of stimuli and underlies cue-reactivity. Few studies have systematically examined cue-induced reactivity over prolonged abstinence periods and none have examined the association between DA signaling and cue-induced reactivity and the relationship to relapse in human smokers. The overall goal of this proposal is to uncover the molecular mechanisms underlying tobacco smoking withdrawal, and the relationship to withdrawal-related behaviors (i.e., cognitive function, mood, cue-reactivity) and relapse. We now have the tools to probe both ACh and DA neurotransmission in the human brain with positron emission computed tomography (PET). We will measure physostigmine-induced elevations in synaptic ACh with [18F]Flubatine and amphetamine-induced DA release with [11C]PHNO, within-subject, in nonsmokers and in tobacco smokers during early and prolonged withdrawal. Further, we will investigate whether alterations in ACh and DA levels differentially affect cognitive function (attention, concentration, cue reactivity) and mood in smokers vs. nonsmokers and whether changes in synaptic ACh or DA are predictive of success in maintaining abstinence for up to 6-8 weeks. If there are predictive relationships between ACh and DA function, cognitive domains and relapse, the cognitive measures may ultimately serve as biomarkers of the imaging measures to direct individualized treatment strategies toward restoration of the cholinergic or dopaminergic systems based on the identified impairments. An elucidation of the mechanisms underlying tobacco smoking withdrawal may ultimately lead to novel and more targeted treatment, and thus have a major impact on public health.