Molecular studies aimed at modifying ligand-gated ion channel function in primary neuronal cultures have been hampered by the limited efficacy of gene transfer techniques. Although viruses can effectively deliver genes to neurons, their preparation requires a substantial effort and their usefulness in delivering multiple subunits of a single protein has not been well established. In this study, we have successfully cotransfected mammalian expression plasmids for all subunits of the muscle nicotinic acetylcholine receptor (nAChR), along with a plasmid containing green fluorescent protein (GFP), into rat forebrain neurons using a calcium phosphate method. Immunostaining experiments and cell counting showed that the nAChR were successfully expressed in approximately 1% of the neurons in the culture. The functionality of the expressed receptors was demonstrated electrophysiologically as carbachol-induced currents were present in nearly 90% of the GFP-positive neurons tested. These currents could be completely inhibited by the irreversible antagonist alpha-cobratoxin. Surprisingly, no currents were obtained in any of the GFP-negative cells tested. These results demonstrate that gene delivery of complex, multi-subunit proteins can be successfully performed in cultured neurons, thereby opening new strategies for the pharmacological study of ligand-gated ion channels.