POTASSIUM channels are highly selective and allow the rapid flux of potassium ions through their pore1. Several studies have implicated the H5 (P or SS1-SS2) segment2,3 as part of the pore in voltage-gated ion channels4-10. The proposal that H5 spans at least 80% of the electric potential drop across the K+ channel pore5,11 is based on altered internal tetraethylammonium sensitivity arising from mutations of H5 residues that are 100% conserved among K+ channels having differing sensitivity to tetraethylammonium5-7,12,13. Here we report that the S6 segment is also involved in K+ ion permeation and in governing the sensitivity to internal tetraethylammonium and barium. Transplanting the S6 segment of NGK2 into Shaker causes this S6 chimaera to adopt the single-channel conductance and sensitivity to internal tetraethylammonium and barium ions from the NGK2 channel. The differences between NGK2 and Shaker in external tetraethylammonium sensitivity, but not single-channel conductance, can be attributed to the differences in their H5 sequences. Three non-conserved S6 residues have been found to affect either single-channel conductance or internal tetraethylammonium sensitivity.