Evidence is now substantial that membrane segments besides H-5 contribute to the pore of K+ channels. We found that substitution of the H-5 region of Shaker B (ShB) with the corresponding sequence of NGK2 expressed channels which retained the single channel K+ conductance (g(K+)) of the host ShB channel. A reverse chimera with ShB H-5 region transplanted into NGK2 also retained the g(K+) of the host NGK2. Point mutations V443L+T449Y in ShB H-5 converted internal tetraethylammonium (TEA) affinity to NGK2 values, and T449Y converted external TEA affinity and Rb+ conductance (g(Rb+)) to NGK2 values. In ShB, exchanging a short stretch of 9 amino acids located just past the transmembrane segment referred to as S-6, post-S-6, produced a large increase in g(K+) with no effect on internal or external TEA blockade. Within S-6, 3 important residues for internal TEA blockade were identified. Thus, H-5 determines external TEA blockade and both H-5 and S-6 may determine internal TEA blockade, but neither H-5 nor S-6 alone restored the donor g(K+). However, chimeric channels in which H-5, S-6, and post-S-6 were exchanged transferred g(K+) of NGK2 to ShB or the g(K+) of ShB to NGK2. Thus, contributions from H-5, S-6, and its cytoplasmic extension post-S-6 make the pore of voltage-dependent Shaker K+ channels a polysegmental mosaic structure.