A family of genes encoding neuronal acetylcholine receptor (AChR) subunits has been identified and cloned from vertebrates. Expression studies have implied that as few as one or two kinds of subunits may be sufficient to construct neuronal AChRs and that multiple pair-wise combinations of the gene products are capable of generating functional receptors. We show here that a class of AChRs with a predominantly synaptic location on neurons contains receptors having at least three types of subunits and that the subunits are encoded by the alpha 3, beta 4, and alpha 5 AChR genes. In addition, we show that a class of extrasynaptic AChRs on the same neurons contains the alpha 7 subunits but lacks the alpha 3, beta 4, and alpha 5 subunits. The results demonstrate that native AChRs on neurons are more complex in composition than previously appreciated and suggest that constraints on subunit interactions limit the kinds of receptor species produced.