Cys-loop receptors are pentameric ligand-gated ion channels (pLGICs) that mediate fast synaptic transmission. Here functional pentameric assembly of truncated fragments comprising the ligand-binding N-terminal ectodomains and the first three transmembrane helices, M1-M3, of both the inhibitory glycine receptor (GlyR) alpha1 and the 5HT(3)A receptor subunits was found to be rescued by coexpressing the complementary fourth transmembrane helix, M4. Alanine scanning identified multiple aromatic residues in M1, M3 and M4 as key determinants of GlyR assembly. Homology modeling and molecular dynamics simulations revealed that these residues define an interhelical aromatic network, which we propose determines the geometry of M1-M4 tetrahelical packing such that nascent pLGIC subunits must adopt a closed fivefold symmetry. Because pLGIC ectodomains form random nonstoichiometric oligomers, proper pentameric assembly apparently depends on intersubunit interactions between extracellular domains and intrasubunit interactions between transmembrane segments.