The present study was undertaken to investigate the effect of doxorubicin (DX) on secretion of catecholamines (CA) evoked by ACh, high K⁢, DMPP and McN-A-343 from the isolated perfused rat adrenal gland and to establish the mechanism of its action. DX (10⁣7∼10⁣6 M) perfused into an adrenal vein for 60 min produced relatively dose- and time-dependent inhibition of CA secretory responses evoked by ACh (5.32⁓10⁣3 M), DMPP (10⁣4 M) and McN-A-343 (10⁣4 M). However, lower dose of DX did not affect CA secretion by high K⁢ (5.6⁓10⁣2 M), but its higher doses depressed time-dependently CA secretion evoked by high K⁢. DX itself did also fail to affect basal CA output. In adrenal glands loaded with DX (3⁓10⁣7 M), CA secretory responses evoked by Bay-K-8644, an activator of L-type Ca2⁢ channels and cyclopiazonic acid, an inhibitor of cytoplasmic Ca2⁢-ATPase were time-dependently inhibited. Furthermore, daunorubicin (3⁓10⁣7 M), given into the adrenal gland for 60 min, attenuated CA secretory responses evoked by ACh, high K⁢, DMPP and McN-A-343. Taken together, these results suggest that DX causes relatively dose- and time-dependent inhibition of CA secretory responses evoked by stimulation of cholinergic (both nicotinic and muscarinic) receptors from the isolated perfused rat adrenal gland. However, lower dose of DX did not affect CA secretion by high K⁢, and higher doses of DX reduced time-dependently CA secretion of high K⁢. It is thought that these effects of DX may be mediated by inhibiting both influx of extracellular calcium into the rat adrenomedullary chromaffin cells and intracelluar calcium release from the cytoplasmic store. Also, there was no difference in the mode of action between DX and daunorubicin in rat adrenomedullary CA secretion.