Aldosterone secretion from the adrenal glands is negatively controlled by atrial natriuretic hormone (ANP) within the physiological range. ANP antagonizes the secretagogue action of K⁺ and angiotensin II by decreasing intracellular Ca²⁺ concentrations. In isolated adrenal glomerulosa cells ANP was found to activate a charybdotoxin-sensitive K⁺ conductance, suggesting that ANP may antagonize the secretagogue action of Ca²⁺-mobilizing signals via opening of Ca²⁺-dependent maxi-K⁺ (BK) channels. To test this hypothesis, we characterized the regulation of aldosterone secretion in isolated adrenal glands from wild-type and BK-/- mice. Tissue slices were obtained from acutely removed adrenal glands and incubated in modified medium 199 at 37°C. In wild-type mice, increasing extracellular K⁺ concentrations from 4.5 to 7.5 mmol/l stimulated aldosterone secretion by ~100%. Additional incubation with ANP inhibited aldosterone release in a dose-dependent fashion with a half maximal inhibition at ~10 nmol/l. Both K⁺-dependent stimulation and ANP-mediated inhibition were entirely preserved in adrenal slices from BK-/- mice. These results demonstrate that ANP can inhibit aldosterone secretion independent of functional BK channels.