The effects of KN62 on aldosterone secretion have been studied using an angiotensin II (AII)- and K+-responsive human adrenocortical tumor cell line (H295R). Basal aldosterone secretion (measured by RIA) was 0.57 ± 0.22 pmol/mg protein.h. The physiological agonists AII (10 nM) and K+ (14 mM) increased aldosterone secretion by 6.9- and 5.0-fold, respectively. Aldosterone secretion was also stimulated by dibutyryl cyclic AMP (dbcAMP, 1 mM, 10.3-fold over basal). Nifedipine dose-dependently inhibited K+- and AII-stimulated aldosterone secretion. In contrast, dbcAMP-stimulated secretion was relatively insensitive to this agent (26.8% inhibition at 1 μM nifedipine). K+- and AII-stimulated aldosterone production was also dose-dependently inhibited by KN62, which produced 93.9% and 82.3% inhibition at 10 μM KN62 (both p<0.01). In order to test the specificity of KN62 in H295R cells, its effects on various other steroidogenic agonists were assessed. KN62 dose-dependently inhibited aldosterone secretion stimulated by dbcAMP, 22-hydroxycholesterol and pregnenolone. In addition, KNO4, a derivative of KN62 which is not a potent inhibitor of CaM Kinase II, exhibited a similar pattern of inhibition. These data confirm the requirement for extracellular Ca2+ in the stimulation of human adrenocortical cell aldosterone secretion by AII and K+. However, the non-specific inhibitory effects of KN62 in H295R cells limit the usefulness of this agent as a tool for investigations of the involvement of CaM kinase II in adrenocortical steroidogenesis.