Assembly of ionotropic neurotransmitter receptors typified by acetylcholine receptors (AChRs) is thought to be directed by an N-terminal extracellular domain of a subunit. Consistent with this hypothesis, chimeras with the delta subunit N-terminal domain fused to the rest of the gamma subunit can substitute for delta, but not gamma, subunits during AChR assembly. However, chimeras with the gamma subunit N-terminal domain fused to the rest of the delta subunit cannot substitute for gamma or delta subunits during assembly. Furthermore, expression of this chimera with the four wild-type subunits prevents the formation of alpha-bungarotoxin (Bgt) binding sites. Instead of AChR pentamers, complexes are assembled containing only the chimera and either alpha or beta subunits. Based on the results of additional gamma-delta chimeras, there are at least two different regions within the C-terminal half of the chimera required for the dominant-negative effect. Our results indicate that the N-terminal domain of the gamma subunit mediates the initial subunit associations, whereas signals in the C-terminal half of the subunit are required for subsequent subunit interactions.