We tested the hypothesis that the folding, assembly and insertion of neuronal nicotinic receptors are critically dependent on the host cell line. We used recombinant adenoviruses encoding either the rat alpha7, alpha4 or beta2 subunits in which expression of the subunit is controlled by a tetracycline-dependent promoter to screen five cell lines (GH4C1, SH-EP1, CV1, SN-56, and CHO-CAR). All five lines do not express detectable nicotinic receptor but do express receptor for human adenovirus, and all expressed mRNA for alpha7, alpha4 and beta2 subunits when infected with viruses. Each cell line expressed varying levels of alpha4beta2 receptors that bound [3H]cytisine, but only the GH4C1 and SH-EP1 cell lines expressed either surface or internal alpha7 receptors that bound [125I]alpha-bungarotoxin ([125I]alpha-BGT). All five cell lines expressed a 60 kDa protein immunoblotted by anti-alpha7 antibodies when infected with the alpha7 virus, presumably representing unassembled alpha7 subunits. In addition, GH4C1 cells expressed over 10-fold more surface alpha7 receptor than SH-EP1 cells, even though the total alpha7 receptor in the two cell lines was similar. Sedimentation experiments indicate that SH-EP1 cells only partially assemble alpha7 receptors compared with GH4C1 cells and control alpha7 from rat brain. These data suggest that not only is surface alpha7 receptor expression a multistep process, but that each step may involve cell-specific assembly factors.