Human nicotinic acetylcholine receptor (nAChR) alpha7 subunits were stably and heterologously expressed in native nAChR-null SH-EP1 human epithelial cells. Immunofluorescence staining shows alpha7 subunit protein expression in virtually every transfected cell. Microautoradiographic analysis identifies 125I-labeled alpha-bungarotoxin (I-Bgt) binding sites corresponding to human alpha7 (halpha7)-nAChRs on the surface of most cells. I-Bgt binds to halpha7-nAChRs in membrane fractions with a typical apparent K(D) value of approximately 5 nM and B(max) value of approximately 1 pmol/mg membrane protein, and 62% of these sites are expressed on the cell surface. Function of heterologously expressed halpha7-nAChRs is evident as rapid, transient inward current responses to (-)-nicotine. Nicotine treatment of transfected cells produces dose- and time-dependent increases (up to approximately 100%) in numbers of I-Bgt binding sites. Epibatidine is a useful ligand for studies of nAChRs containing alpha3 or alpha4 subunits (K(D) values of about 100 or 10 pM, respectively). halpha7-nAChRs expressed in transfected SH-EP1 cells also exhibit picomolar affinity binding of 3H-labeled epibatidine (K(D) value of approximately 0.6 nM). Studies of several forms of native or heterologously expressed rat or human alpha7-nAChRs confirm high-affinity and mutually exclusive interaction with both epibatidine and alpha-bungarotoxin. Rank order potencies for drugs acting to compete for binding of either radioligand are similar (methyllycaconitine > dimethylphenyl-piperazinium > nicotine approximately cytisine > carbamylcholine approximately D-tubocurarine). These results demonstrate that transfected SH-EP1 cells are excellent models for studies of heterologously expressed, human alpha7-nAChRs that exhibit ligand binding and functional properties like native alpha7-nAChRs and that epibatdine is useful as a probe for human alpha7-nAChRs.