Structural models of cysteine (Cys)-loop receptors based on homology with the Torpedo marmorata nicotinic acetylcholine (nACh) receptor infer the existence of cytoplasmic portals within the conduction pathway framed by by helical amphipathic regions (termed MA-helices) of adjacent intracellular M3-M4 loops. Consistent with these models, two arginine residues (R436 and R440) within the MA-helix of 5-HT3A receptors act as rate limiting determinants of single-channel conductance (?). However, there is little conservation in primary amino acid sequences across the cytoplasmic loops of Cys-loop receptors, limiting confidence in the fidelity of this particular aspect of the 5-HT3A receptor model. We probed the majority of residues within the MA-helix of the human 5-HT3A subunit using alanine- and arginine-scanning mutagenesis and the substituted-cysteine accessibility method to determine their relative influences upon ?. Numerous residues, prominently those at the 435, 436, 439 and 440 positions were found to markedly influence ?. This approach yielded a functional map of the 5-HT3A receptor portals which agrees well with the homology model.