Aim: To study the effect of arecoline, an alkaloid isolated from Areca catechu, on the secretion of catecholamines (CA) evoked by cholinergic agonists and the membrane depolarizer from isolated perfused rat adrenal gland. Methods: Adrenal glands were isolated from male Sprague‐Dawley rats. The adrenal glands were perfused with Krebs bicarbonate solution by means of a peristaltic pump. The CA content of the perfusate was measured directly using the fluorometric method. Results: Arecoline (0.1–1.0 mmol/L) perfused into an adrenal vein for 60 min produced dose‐ and time‐dependent inhibition in CA secretory responses evoked by acetylcholine (ACh) (5.32 mmol/L), 1.1‐dimethyl‐4‐phenyl piperazinium iodide (DMPP) (100 μmol/L for 2 min) and 3‐(m‐choloro‐phenyl‐carbamoyl‐oxy)‐2‐butynyl trimethyl ammonium chloride (McN‐A‐343) (100 μmol/L for 2 min). However, lower doses of arecoline did not affect CA secretion of high K+ (56 mmol/L); higher doses greatly reduced CA secretion of high K+. Arecoline also failed to affect basal catecholamine output. Furthermore, in adrenal glands loaded with arecoline (0.3 mmol/L), CA secretory response evoked by Bay‐K‐8644 (10 μmol/L), an activator of L‐type Ca2+ channels, was markedly inhibited, whereas CA secretion by cyclopiazonic acid (10 μmol/L), an inhibitor of cytoplasmic Ca2+‐ATPase, was not affected. Nicotine (30 μmol/L), which was perfused into the adrenal gland for 60 min, however, initially enhanced ACh‐evoked CA secretory responses. As time elapsed, these responses became more inhibited, whereas the initially enhanced high K+‐evoked CA release diminished. CA secretion evoked by DMPP and McN‐A‐343 was significantly depressed in the presence of nicotine. Conclusion: Arecoline dose‐dependently inhibits CA secretion from isolated perfused rat adrenal gland evoked by activation of cholinergic receptors. At lower doses arecoline does not inhibit CA secretion through membrane depolarization, but at larger doses it does. This inhibitory effect of arecoline may be mediated by blocking the calcium influx into the rat adrenal medullary chromaffin cells without the inhibition of Ca2+ release from the cytoplasmic calcium store. There seems to be a difference in the mode of action of nicotine and arecoline in rat adrenomedullary CA secretion.