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Amiloride-sensitive cationic channels are present in the apical membrane of porcine thyroid cells in primary culture. An amiloride-sensitive (K0.5= 150 ± 28 nM where K0.5 is the concentration of unlabelled ligand which reduces the specific binding of the same labelled ligand by 50%) 22Na+-flux component (Km for Na+ at 18 mM) has been identified which was also blocked by the potent amiloride derivative phenamil (K0.5= 47 ± 21 nM). The most potent inhibitor of Na+/H+ exchange, ethylisopropyl-amiloride, hardly inhibited this 22Na+-influx component at a concentration of 21 μM. Amiloride binding sites were characterized using [3H]phenamil. The tritiated ligand binds to a single family of binding sites in thyroid membranes with a Kd value of 50 ± 10 nM and a maximal binding capacity of 5 ± 1 pmol/mg protein. Patch-clamp experiments have directly demonstrated the existence of a phenamil- and amiloride-sensitive cationic channel, with a conductance of 2.6 pS, which is permeable to sodium, but not very selective (PNa-/PK+= 1.2). This channel is an important element in the regulation of the resting membrane potential of thyroid cells.