We previously reported the partial purification and characterization of a toxic substance (sea urchin toxin) isolated from the pedicellariae of the sea urchin Toxopneustes pileolus (Nakagawa and Kimura, Jpn J Pharmacol 32: 966-968, 1982). In the present study, we examined the effect of sea urchin toxin on catecholamine secretion and synthesis in cultured bovine adrenal medullary cells. Sea urchin toxin inhibited the secretion of catecholamines stimulated by carbachol and nicotine but not by veratridine or a high concentration of K+. The toxin inhibited the carbachol-evoked influx of 22Na+ and 45Ca2+ at concentrations similar to those for catecholamine secretion. The inhibition of catecholamine secretion by sea urchin toxin was not overcome by increasing the concentration of carbachol. Preincubation of cells with the toxin caused a time-dependent inhibition in the secretion stimulated by carbachol even when the toxin was removed from the incubation medium. The toxin suppressed catecholamine synthesis and tyrosine hydroxylase activity in carbachol-stimulated cells. In addition, sea urchin toxin inhibited [3H]phencyclidine binding to adrenal medullary cells whereas it did not alter cyclic GMP accumulation caused by muscarine. Further purified fractions from sea urchin toxin by concanavalin A affinity column chromatography also inhibited carbachol-evoked secretion of catecholamines. These results suggest that sea urchin toxin inhibits carbachol-enhanced secretion and synthesis of catecholamines by suppression of nicotinic acetylcholine receptor-mediated Na+ influx and subsequent Ca2+ influx in cultured adrenal medullary cells.