CONTEXT: Varenicline, an effective smoking cessation medication, functions as an α4ß2 nicotinic acetylcholine receptor partial agonist. It indirectly affects the dopaminergic reward system by reducing withdrawal symptoms during abstinence and by decreasing the reinforcement received from nicotine while smoking. We hypothesize that varenicline would have a third mechanism to blunt responses to smoking cues in the reward-related ventral striatum and medial orbitofrontal cortex and would be associated with a reduction in smoking cue­elicited craving. DESIGN: A laboratory model of conditioned responding and arterial spin-labeled perfusion functional magnetic resonance imaging, a biomarker of regional brain activity, was used to test our hypothesis. Perfusion functional magnetic resonance imaging is quantitative and stable across time, facilitating the measurement of medication-induced neural modifications in the brain in response to a challenge (smoking cue exposure) and in the brain in the resting condition (without provocation). Smokers were imaged during rest and during smoking cue exposure before and after a 3-week randomized placebo-controlled medication regimen. Subjects were nonabstinent to explicitly examine the effects of varenicline on cue reactivity independent of withdrawal. SETTING: Center for the Study of Addictions, University of Pennsylvania, Philadelphia. Subjects Subjects were nicotine-dependent smokers who responded to advertisements placed on local radio and Listservs to participate in a medication-related research study that specifically stated "this is not a Quit Smoking Study" and "smokers may be contemplating but not currently considering quitting." RESULTS: Prerandomization smoking cues vs nonsmoking cues activated the ventral striatum and medial orbitofrontal cortex (t = 3.77) and elicited subjective reports of craving (P = .006). Craving reports correlated with increased activity in the posterior cingulate (t = 4.11). Administration of varenicline diminished smoking cue­elicited ventral striatum and medial orbitofrontal cortex responses (t values from ­3.75 to ­5.63) and reduced self-reported smoking cue­elicited craving, whereas placebo-treated subjects exhibited responses similar to those observed prior to randomization. Varenicline-induced activation of lateral orbitofrontal cortex in the brain at rest (t = 5.63) predicted blunting of smoking cue responses in the medial orbitofrontal cortex (r = ­0.74). CONCLUSIONS: Varenicline's reciprocal actions in the reward-activated medial orbitofrontal cortex and in the reward-evaluating lateral orbitofrontal cortex underlie a diminished smoking cue response, revealing a distinctive new action that likely contributes to its clinical efficacy.