We prepared concatamers of alpha4 and beta2 subunits for human nicotinic acetylcholine receptors (AChRs), in which the C terminus of alpha4 was linked to the N terminus of beta2, or vice versa, via a tripeptide sequence repeated 6 or 12 times, and expressed them in Xenopus oocytes. Linkage did not substantially alter channel amplitude or channel open-duration. Linkage at the C terminus of alpha4 prevented AChR potentiation by 17-beta-estradiol by disruption of its binding site. Assembly of AChRs from concatamers was less efficient, but function was much more efficient than that of unlinked subunits. With both linked and free subunits, greater ACh-induced currents per surface AChR were observed with the (alpha4)3(beta2)2 stoichiometry than with the (alpha4)2(beta2)3 stoichiometry. The (alpha4)3(beta2)2 stoichiometry exhibited much lower ACh sensitivity. When concatamers were expressed alone, dipentameric AChRs were formed in which the (alpha4)2(beta2)3 pentamer was linked to the (alpha4)3(beta2)2 pentamer. Dipentamers were selectively expressed on the cell surface, whereas most monopentamers with dangling subunits were retained intracellularly. Coexpression of concatamers with monomeric beta2, beta4, or alpha4 subunits resulted in monopentamers, the stoichiometry of which was determined by the free subunit added. Linkage between the C terminus of beta2 and the N terminus of alpha4 favored formation of ACh-binding sites within the concatamer, whereas linkage between the C terminus of alpha4 and the N terminus of beta2 favored formation of ACh-binding sites between concatamers. These protein-engineering studies provide insight into the structure and function of alpha4beta2 AChRs, emphasizing the functional differences between alpha4beta2 AChRs of different stoichiometries.