To better understand smoking cessation, we examined the actions of varenicline (Chantix) during long-term nicotine exposure. Varenicline reduced nicotine upregulation of ?4?2-type nicotinic receptors (?4?2Rs) in live cells and neurons, but not for membrane preparations. Effects on upregulation depended on intracellular pH homeostasis and were not observed if acidic pH in intracellular compartments was neutralized. Varenicline was trapped as a weak base in acidic compartments and slowly released, blocking 125I-epibatidine binding and desensitizing ?4?2Rs. Epibatidine itself was trapped; 125I-epibatidine slow release from acidic vesicles was directly measured and required the presence of ?4?2Rs. Nicotine exposure increased epibatidine trapping by increasing the numbers of acidic vesicles containing ?4?2Rs. We conclude that varenicline as a smoking cessation agent differs from nicotine through trapping in ?4?2R-containing acidic vesicles that is selective and nicotine-regulated. Our results provide a new paradigm for how smoking cessation occurs and suggest how more effective smoking cessation reagents can be designed.