Nicotine causes changes in brain nicotinic acetylcholine receptors (nAChRs) during smoking that initiate addiction. Nicotine-induced upregulation is the long-lasting increase in nAChR radioligand binding sites in brain resulting from exposure. The mechanisms causing upregulation are not established. Many different mechanisms have been reported with the assumption that there is a single underlying cause. Using live rat cortical neurons, we examined for the first time how exposure and withdrawal of nicotine shape the kinetics of native ?4?2-containing nAChR upregulation in real time. Upregulation kinetics demonstrates that at least two different mechanisms underlie this phenomenon. First, a transient upregulation occurs that rapidly reverses, faster than nAChR degradation, and corresponds to nAChR conformational changes as assayed by conformational-dependent, subunit-specific antibodies. Second, a long-lasting process occurs correlating with increases in nAChR numbers caused by decreased proteasomal subunit degradation. Previous radioligand binding measurements to brain tissue have measured the second process and largely missed the first. We conclude that nicotine-induced upregulation is composed of multiple processes occurring at different rates with different underlying causes.