Recent evidence suggests that in addition to A4B2 and A3-containing nicotinic receptors, A6-containing receptors are present in midbrain dopaminergic neurons and involved in the nicotine reward pathway. Using heterologous expression, we found that A6B2, like A3B2 and A4B2 receptors, formed high-affinity, epibatidine–binding complexes that are pentameric, trafficked to the cell surface and produced acetylcholine-evoked currents. Chronic nicotine exposure upregulated A6B2 receptors with differences in upregulation time course and concentration-dependence compared to A4B2 receptors, the predominant high-affinity nicotine binding site in brain. The A6B2 receptor upregulation required higher nicotine concentrations than for A4B2 but was 10-fold faster and more comparable to that of A3B2. Our data suggest that nicotinic receptor upregulation is subtype specific such that A6/A3-containing receptors upregulate in response to transient, high nicotine exposures while sustained, low nicotine exposures upregulate A4B2 receptors.