Many nicotinic acetylcholine receptor (AChR) subunits are known to be co-expressed with the alpha6 subunit in neurons. Because alpha6beta4 AChRs assemble inefficiently and alpha6beta2 AChRs not at all, more complex mixtures of human subunit cDNAs were tested for their abilities to form functional AChRs when expressed in Xenopus oocytes. alpha6beta4beta3 AChRs produced the largest and most consistent responses. alpha6alpha3beta2 AChRs exhibited reduced potency for ACh and increased potency and efficacy for nicotine compared to alpha3beta2 AChRs, but similar resistance to functional inactivation after prolonged exposure to nicotine. alpha6alpha4beta2 AChRs differed little in potency or efficacy for ACh or nicotine compared to alpha4beta2 AChRs, and had similarly high sensitivity to inactivation by prolonged exposure to nicotine. Co-expression of alpha6 and beta2 cRNAs resulted in large numbers of (3)H-epibatidine binding sites in the form of large aggregates but not in functional pentameric AChRs. Co-expression of alpha6, beta2, and alpha5 resulted in assembly of some functional pentameric AChRs. Chimeras with the large extracellular domain of alpha6 and the rest from either alpha3 or alpha4 efficiently formed functional AChRs. Thus, the extracellular domain of alpha6 efficiently assembles with beta2 to form ACh binding sites, but more C-terminal domains cause difficulties in forming pentameric AChRs. Chimeric alpha6/alpha3 and alpha6/alpha4 AChRs containing either beta2 or beta4 subunits were blocked by alpha-conotoxin MII which had previously been reported to be specific for alpha3beta2 AChRs.