To elucidate the mechanism of protamine-induced hypotension, we examined the effects of protamine on catecholamine secretion in bovine adrenal medullary cells and on the serum norepinephrine in the rat. 1) in bovine adrenal medullary cells in culture, protamine at concentrations of 10 to 100 micrograms/ml inhibited catecholamine secretion stimulated by carbachol. The inhibitory effect of protamine was diminished by heparin at concentrations of 3.5 to 14 U/ml. Protamine suppressed carbachol-stimulated 22Na+ influx and 45Ca++2 influx at a concentration similar to that which inhibited catecholamine secretion. Protamine (10-100 micrograms/ml) also inhibited veratridine-induced 22Na+ influx and 45Ca++ influx and 56 mM K(+)-evoked 45Ca++ influx. The inhibition of these ion influxes by protamine was closely correlated with that of catecholamine secretion. 2) In rats, i.v. administration of protamine (10 mg/kg) attenuated the arterial blood pressure and the serum norepinephrine. There was a high correlation (r = 0.96) between the serum norepinephrine and the arterial blood pressure in protaminetreated rats. Furthermore, pretreatment with heparin (1000 U/kg) abolished the protamine-induced decreases in arterial blood pressure and serum norepinephrine. Because protamine seems to inhibit catecholamine secretion by interfering with Na+ influx and Ca++ influx to adrenal medullary cells, the protamine-induced hypotension may be, at least in part, due to inhibition of norepinephrine release and ion channel activities of sympathetic nerve terminals in rats.