Nicotinic acetylcholine (nACh) and 5-hydroxytryptamine type 3 (5-HT(3)) receptors are cation-selective ion channels of the pentameric ligand-gated ion channel (pLGIC) superfamily. Multiple lines of evidence adduced over the last 30 years indicate that the lining of the channel of such receptors is formed by the alpha-helical second transmembrane (TM2) domain and flanking sequences contributed by each of the five subunits present within the receptor complex. Specific amino acid residues within, and adjacent to, the TM2 domain influence single channel conductance, ion selectivity, and other aspects of receptor function that include gating and desensitization. However, more recent work has revealed important structural determinants of single channel conductance and ion selectivity that are not associated with the TM2 domain. Direct experimental evidence indicates that the intracellular domain of eukaryotic pLGICs, in particular a region of the loop linking TM3 and TM4 termed the membrane-associated (MA) stretch, exerts a strong influence upon ion channel biophysics. Moreover, recent computational approaches, complemented by experimentation, implicate the extracellular domain as an additional important determinant of ion conduction. This brief review describes how our knowledge of ion conduction and selectivity in cation-selective pLGICs has evolved beyond TM2.