Previously, a rat brain cDNA was reported that was designated alpha6 because of its homology with nicotinic acetylcholine receptor (AChR) alpha subunits, being especially similar to alpha3, but no acetylcholine-gated cation channels were detected when it was expressed in Xenopus laevis oocytes alone or in combination with other known rat AChR subunits. We cloned chicken alpha6 and human beta4 AChR subunits and tested for acetylcholine-gated cation channels with alpha6 by expression in X. laevis oocytes alone or in pairwise combination with chicken alpha3, beta2, or beta4 or with human alpha3, beta2, or beta4 AChR subunits. Chicken alpha6 formed detectable functional AChRs only when expressed together with the human beta4 subunit. The alpha6beta4 AChR-mediated currents show strong inward rectification and dependence on extracellular Ca2+. It exhibited a distinct pharmacological profile with an EC50 value of 28 microM for acetylcholine, 24 nM for (+)-epibatidine, 6.6 microM cytisine, and 15 microM 1,1-dimethyl-4-phenylpiperazinium. Both cytisine and 1,1-dimethyl-4-phenylpiperazinium behaved as partial (approximately 30%) agonists. Remarkably, nicotine (EC50 = 22 microM) was an even weaker partial agonist (approximately 18%) and had a relatively long-lasting inhibitory effect. Coexpression of the previously cloned rat alpha6 subunit with the human the beta4 subunit also resulted in functional alpha6beta4 AChRs with properties resembling those of the chicken/human alpha6beta4 AChRs. Therefore, alpha6 can function as part of AChRs with unusual pharmacological properties.