The organic cation 2,4,6-triaminopyrimidinium (TAP) specifically inhibits Na+ passive permeation (PNa) across gallbladder, small intestine, and choroid plexus without detectable effect on the Cl(-) permeability, indicating that Na+ and Cl(-) follow different permeation pathways. In bullfrog gallbladder, where it was examined in greater detail, the effect of TAP was shown to be: (a) completely reversible, (b) due only to the protonated form of 2,4,6-triaminopyrimidine, (c) effective when added to either one or both sides of the membrane (the rate limiting for the delay in the response being the diffusion through the unstirred layers), and (d) exhibiting a typical saturation kinetics, best fitted with the parameters "Km" = 2.6 mM and maximal effect = 100% inhibition. These data, along with the fact that the PNa blocking action of chemical analogs of TAP increases with their ability to donate protons to form hydrogen bonds, suggest that TAP blocks the cation permeation of the channels by strongly associating, via hydrogen bonds, with the anionic ligands within the channel.