Abstract Tobacco continues to be widely used world-wide, primarily via cigarette smoking, and is the leading cause of preventable deaths in the United States. Tobacco use is driven by nicotine addiction, which starts by nicotine binding to high-affinity nicotine binding sites in brain. 80-90% of the high-affinity sites are located on α4β2-type nicotinic acetylcholine receptors (α4β2Rs). Prolonged nicotine exposure increases high-affinity α4β22R binding sites in brain, a process termed “upregulation”, linked to craving and withdrawal in nicotine addiction. This proposal is based on our recent discovery that α4β2R ligands that are weak bases, such as the smoking cessation reagent varenicline (Chantix), can be selectively trapped in α4β2R-containing acidic vesicles of cells and neurons. Slow release of trapped varenicline reduces the effects of nicotine upregulation. Selective trapping is further regulated by nicotine upregulation, which increases the numbers of α4β2R-containing acidic vesicles. Nicotine, also a weak base, is not trapped because its ligand pKa and affinity for α4β22Rs is lower than that of varenicline. These results provide a new paradigm for how varenicline causes smoking cessation. They also provide new information about the potential cellular distribution of α4β22R PET probes, all of which are weak bases. Like varenicline and nicotine, different α4β2R PET probes have different ligand pKas and affinities for α4β2Rs, which explains differences in kinetics, displaceable binding by varenicline and nicotine, non-displaceable binding and metabolism. While a number of studies have used PET probes specific for α4β22R high-affinity binding sites in brain, these studies are complicated by the interpretations of the binding and binding kinetics especially when nicotine and/or varenicline are present. Using our concept about the trapping of α4β2R weak base ligands in intracellular acidic vesicles, we will develop new cellular and whole models of PET probe kinetics that take into account α4β2R ligand trapping in acidic vesicles. There is the potential of wider application of the PET methods that will be developed in this application, since for α4β2R PET imaging is currently underway in a number of brain disorders. The goals of this proposal are to examine how our discovery of the trapping of weak base α4β2R ligands in acid vesicles affects the imaging of α4β2Rs using PET probes and to use PET probe imaging to examine how nicotine causes α4β2R upregulation and how varenicline alters upregulation.