1 The aim of the present study was to investigate the direct effects and action mechanisms of digitalis on the production of corticosterone in rat adrenocortical cells. 2 Male rats were challenged with digoxin (1 mg ml À1 kg À1 ) in the presence or absence of adrenocorticotropin (ACTH, 5 mg ml À1 kg À1 ) administered by intravenous injection to the right jugular vein. Blood samples were collected at 0, 30, 60, and 120 min following the challenge. The concentration of corticosterone in the rat plasma samples was measured by radioimmunoassay. 3 Zona fasciculata-reticularis (ZFR) cells in male rats were prepared and then incubated with or without digoxin or digitoxin in the presence or absence of ACTH (10 À9 M), forskolin (10 À7 M), 8-bromo-cyclic 3 0 : 5 0 -adenosine monophosphate (10 À4 M), cyclopiazonic acid (CPA, 10 À5 M), trilostane (10 À6 M), 25-OH-cholesterol (10 À5 M), pregnenolone (10 À5 M), progesterone (10 À5 M), or deoxycorticosterone (10 À5 M) at 371C for 1 h before collection of the media. Corticosterone or pregnenolone levels were measured by radioimmunoassay. 4 A single injection of digoxin did not alter the basal level of plasma corticosterone, but did inhibit the level of plasma corticosterone released in response to ACTH in vivo. 5 Administration of digoxin or digitoxin decreased both spontaneous and ACTH-stimulated release of corticosterone in vitro. 6 Digoxin (10 À7 -10 À5 M) and digitoxin (10 À7 -10 À5 M), but not ouabain (10 À7 -10 À5 M), dosedependently inhibited corticosterone production in response to forskolin and 8-Br-cyclic AMP in rat ZFR cells. 7 Both digoxin (10 À6 -10 À5 M) and digitoxin (10 À6 -10 À5 M) attenuated corticosterone production in response to CPA. 8 Digoxin (10 À5 M) or digitoxin (10 À5 M) inhibited cytochrome P450 side-chain cleavage enzyme (cytochrome P450scc) activity (catalyses conversion of cholesterol to pregnenolone in the presence of trilostane) in rat ZFR cells. 9 The enzyme activity of 11 b-hydroxylase (catalyses conversion of deoxycorticosterone to corticosterone) in ZFR cells was also inhibited by the administration of digoxin (10 À5 M) or digitoxin (10 À5 M). 10 These results together suggest that digoxin and digitoxin decrease the release of corticosterone by acting directly on ZFR cells via a Na þ , K þ -ATPase-independent mechanism involving the inhibition of the activities of adenylyl cyclase, cytochrome P450scc and 11 b-hydroxylase, as well as the functioning of cyclic AMP and intracellular calcium.