Potassium channels are tetrameric proteins that mediate K +-selective transmembrane diffusion. For KcsA, tetramer stability depends on interactions between permeant ions and the channel pore. We have examined the role of pore blockers on the tetramer stability of KirBac1.1. In 150 mM KCl, purified Kir-Bac1.1 protein migrates as a monomer (∼40 kDa) on SDS-PAGE. Addition of Ba2+ (K1/2 ∼ 50 μM) prior to loading results in an additional tetramer band (∼160 kDa). Mutation A109C, at a residue located near the expected Ba2+-binding site, decreased tetramer stabilization by Ba2+ (K1/2 ∼ 300 μM), whereas I131C, located nearby, stabilized tetramers in the absence of Ba 2+. Neither mutation affected Ba2+ block of channel activity (using 86Rb+ flux assay). In contrast to Ba 2+, Mg2+ had no effect on tetramer stability (even though Mg2+ was a potent blocker). Many studies have shown Cd2+ block of K+ channels as a result of cysteine substitution of cavity-lining M2 (S6) residues, with the implicit interpretation that coordination of a single ion by cysteine side chains along the central axis effectively blocks the pore. We examined blocking and tetramer-stabilizing effects of Cd2+ on KirBac1.1 with cysteine substitutions in M2. Cd2+ block potency followed an α-helical pattern consistent with the crystal structure. Significantly, Cd2+ strongly stabilized tetramers of I138C, located in the center of the inner cavity. This stabilization was additive with the effect of Ba2+, consistent with both ions simultaneously occupying the channel: Ba2+ at the selectivity filter entrance and Cd2+ coordinated by I138C side chains in the inner cavity.