To elucidate the mechanism of palytoxin (PTX)-induced Na+ influx, we examined the effect of amiloride, an inhibitor of Na+/H(+)-antiporter, on PTX-induced Na+ influx into cultured bovine adrenal chromaffin cells in relation to its effects on Ca2+ influx and catecholamine secretion. Amiloride dose-dependently inhibited PTX-induced 22Na+ influx, whereas tetrodotoxin (TTX) had no effect. Amiloride also inhibited PTX-induced Na(+)-dependent 45Ca2+ influx and catecholamine secretion. PTX alone did not significantly affect the intracellular pH, but it decreased in the presence of PTX and amiloride. These results indicate that an amiloride-sensitive Na+/H+ exchange mechanism is probably involved in PTX-induced, TTX-insensitive Na+ influx that triggers Ca2+ influx and catecholamine secretion from the cells.