The rat connexin40 gap junction channel is permeable to monovalent cations including tetramethylammonium and tetraethyiammonium ions. Larger tetraalkyammonium (TAA+) ions beginning with tetrabutylammonium (TBA+) reduced KCI junctional currents disproportionately. Ionic blockade by tetrapentylammonium (TPeA+) and tetrahexylammonium (THxA+) ions were concentration- and voltage-dependent and occurred only when TAA+ ions were on the same side as net K+ efflux across the junction, indicative of block of the ionic permeation pathway. The voltage-dependent dissociation constants (Km(Vj)) were lower for THxA+ than TPeA+, consistent with steric effects within the pore. The Km-Vj relationships for TPeA+ and THxA+ were fit with different reaction rate models for a symmetrical (homotypic) connexin gap junction channel and were described by either a one- or two-site model that assumed each ion traversed the entire Vj field. Bilateral addition of TPeA+ ions confirmed a common site of interaction within the pore that possessed identical Km(Vj) values for cis-trans concentrations of TPeA+ ions as indicated by the modeled I-V relations and rapid channel block that precluded unitary current measurements. The TAA+ block of K+ currents and bilateral TPeA+ interactions did not alter Vj-gating of Cx40 gap junctions. N-octyl-tributylammonium and -triethylammonium also blocked rCx40 channels with higher affinity and faster kinetics than TBA+ or TPeA+, indicative of a hydrophobic site within the pore near the site of block.