Bovine adrenal zona fasciculata cells (AZF) express a noninactivating K⁺ current (I_AC) whose inhibition by adrenocorticotropic hormone and ANG II may be coupled to membrane depolarization and Ca²⁺-dependent cortisol secretion. We studied I_AC inhibition by Ca²⁺ and the Ca²⁺ ionophore ionomycin in whole cell and single-channel patch-clamp recordings of AZF. In whole cell recordings with intracellular (pipette) Ca²⁺ concentration ([Ca²⁺]_i) buffered to 0.02 µM, I_AC reached maximum current density of 25.0 ± 5.1 pA/pF (n = 16); raising [Ca²⁺]_i to 2.0 µM reduced it 76%. In inside-out patches, elevated [Ca²⁺]_i dramatically reduced I_AC channel activity. Ionomycin inhibited I_AC by 88 ± 4% (n = 14) without altering rapidly inactivating A-type K⁺ current. Inhibition of I_AC by ionomycin was unaltered by adding calmodulin inhibitory peptide to the pipette or replacing ATP with its nonhydrolyzable analog 5'-adenylylimidodiphosphate. I_AC inhibition by ionomycin was associated with membrane depolarization. When [Ca²⁺]_i was buffered to 0.02 µM with 2 and 11 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), ionomycin inhibited I_AC by 89.6 ± 3.5 and 25.6 ± 14.6% and depolarized the same AZF by 47 ± 8 and 8 ± 3 mV, respectively (n = 4). ANG II inhibited I_AC significantly more effectively when pipette BAPTA was reduced from 11 to 2 mM. Raising [Ca²⁺]_i inhibits I_AC through a mechanism not requiring calmodulin or protein kinases, suggesting direct interaction with I_AC channels. ANG II may inhibit I_AC and depolarize AZF by activating parallel signaling pathways, one of which uses Ca²⁺ as a mediator.