Among ion channels, only the nicotinic-receptor superfamily has evolved to generate both cation- and anion-selective members. Although other, structurally unrelated, neurotransmitter-gated cation channels exist, no other type of neurotransmitter-gated anion channel, and thus no other source of fast synaptic inhibitory signals, has been described so far. In addition to the seemingly straightforward electrostatic effect of the presence (in the cation-selective members) or absence (in the anion-selective ones) of a ring of pore-facing carboxylates, mutational studies have identified other features of the amino-acid sequence near the intracellular end of the pore-lining transmembrane segments (M2) that are also required to achieve the high charge selectivity displayed by native channels1–10. However, the mechanism underlying this subtler effect has remained elusive11 and a subject of much speculation. Here, using single-channel electrophysiological recordings to estimate the protonation state of native ionizable side chains, we show that anion-selective type sequences favour, whereas cation-selective type sequences prevent, the protonation of the conserved, buried basic residues at the intracellular entrance of the pore (the M2 0′ position). We conclude that the, previously unrecognized, tunable charge state of the 0′ ring of buried basic side chains is an essential feature of these channels’ versatile charge-selectivity filter.