Heteromeric a3b4 nicotinic acetylcholine (ACh) receptors (nAChRs) are pentameric ligand-gated cation channels that include at least two a3 and two b4 subunits. They have functions in peripheral tissue and peripheral and central nervous systems. We examined the effects of chronic treatment with menthol, a major flavor additive in tobacco cigarettes and electronic nicotine delivery systems, on mouse a3b4 nAChRs transiently transfected into neuroblastoma-2a cells. Chronic menthol treatment at 500 nM, near the estimated menthol concentration in the brain following cigarette smoking, altered neither the [ACh]response relationship nor Zn 21 sensitivity of ACh-evoked currents, suggesting that menthol does not change a3b4 nAChR subunit stoichiometry. Chronic menthol treatment failed to change the current density (peak current amplitude/cell capacitance) of 100 mM ACh-evoked currents. Chronic menthol treatment accelerated desensitization of 100 and 200 mM AChevoked currents. Chronic nicotine treatment (250 mM) decreased ACh-induced currents, and we found no additional effect of including chronic menthol. These data contrast with previously reported, marked effects of chronic menthol on b2* nAChRs studied in the same expression system. Mechanistically, the data support the emerging interpretation that both chronic menthol and chronic nicotine act on nAChRs in the early exocytotic pathway, and that this pathway does not present a rate-limiting step to the export of a3b4 nAChRs; these nAChRs include endoplasmic reticulum (ER) export motifs but not ER retention motifs. Previous reports show that smoking mentholated cigarettes enhances tobacco addiction; but our results show that this effect is unlikely to arise via menthol actions on a3b4 nAChRs.