Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is linked with high penetrance to several distinct nicotinic receptor (nAChR) mutations. We studied (alpha 4)(3)(beta 2)(2) versus (alpha 4)(2)(beta 2)(3) subunit stoichiometry for five channel-lining M2 domain mutations: S247F, S252L, 776ins3 in alpha 4, V287L, and V287M in beta 2. alpha 4 and beta 2 subunits were constructed with all possible combinations of mutant and wild-type (WT) M2 regions, of cyan and yellow fluorescent protein, and of fluorescent and nonfluorescent M3-M4 loops. Sixteen fluorescent subunit combinations were expressed in N2a cells. Forster resonance energy transfer (FRET) was analyzed by donor recovery after acceptor photobleaching and by pixel-by-pixel sensitized emission, with confirmation by fluorescence intensity ratios. Because FRET efficiency is much greater for adjacent than for nonadjacent subunits and the alpha 4 and beta 2 subunits occupy specific positions in nAChR pentamers, observed FRET efficiencies from (alpha 4)(3)(beta 2)(2) carrying fluorescent alpha 4 subunits were significantly higher than for (alpha 4)(3)(beta 2)(2); the converse was found for fluorescent beta 2 subunits. All tested ADNFLE mutants produced 10 to 20% increments in the percentage of intracellular (alpha 4)(3)(beta 2)(2) receptors compared with WT subunits. In contrast, 24- to 48-h nicotine (1 mu M) exposure increased the proportion of (alpha 4)(3)(beta 2)(2) in WT receptors and also returned subunit stoichiometry to WT levels for alpha 4S248F and beta 2V287L nAChRs. These observations may be relevant to the decreased seizure frequency in patients with ADNFLE who use tobacco products or nicotine patches. Fluorescence-based investigations of nAChR subunit stoichiometry may provide efficient drug discovery methods for nicotine addiction or for other disorders that result from dysregulated nAChRs.