We have used a species difference in E subunits of the acetylcholine receptor (AChR) to investigate regions of the subunit protein that are important in receptor assembly. Upon transient transfection of COS cells, mouse E subunit cDNA is approximately 10 times more effective than that of the rat in supporting expression of surface AChRs when the other subunits are from either mouse or rat. In cells transfected with only a and E subunit cDNAs, the formation of an a& heterodimer, a presumed assembly intermediate, is also less efficient with rat than, with mouse E subunit. By site-directed mutagenesis, we nave found that these differences can be accounted for my 2 amino acid differences in the N-terminal domain at oositions 106 and 115 of the rat and mouse E subunits, Puggesting that the region near these 2 amino acid residues is important for AChR assembly.