The effects on the amiloride-blockable Na+ channel of a family of recently synthesized isothiouronium derivatives were measured in plasma membrane vesicles from rat distal colon. Some of these derivatives act as high-affinity Na(+)-like antagonists on the Na(+)-K(+)-adenosinetriphosphatase. One of the reagents tested, 1-bromo-2,4,6-tris(isothiouronium methyl)-benzene tribromide (Br-TITU), was found to be a potent blocker of the Na+ channel. At neutral pH, Br-TITU rapidly inhibits the channel mediated 22Na+ uptake, with an inhibition constant of 94 +/- 39 nM. The inhibition observed is specific and reversible. 1,3-Dibromo-2,4,6-tris(isothiouronium methyl)benzene tribromide and Br-TITU derivatives with methyl and phenyl substitutions on the isothiouronium moiety were much less effective blockers. Incubation of cells with Br-TITU at alkaline (but not neutral) pH produces irreversible inactivation of channels, possibly due ot covalent modification of a lysine residue. This inactivation can be attenuated by amiloride but not by Na+. Thus Br-TITU may be a useful reagent in identifying essential residues of the channel protein.