Bovine adrenal zona fasciculata cells express background K⁺ channels (I_AC channels) whose activity is potently inhibited by ACTH. In whole-cell patch clamp recordings, it was discovered that the trivalent lanthanides (Ln³⁺s) lanthanum and ytterbium interact with two binding sites to modulate K⁺ flow through these channels. In spite of large differences in ionic radii, these Ln³⁺s inhibited I_AC channels half maximally with IC₅₀s near 50 µM. In addition, these Ln³⁺s blocked and reversed ACTH-mediated inhibition of I_AC K⁺ channels at similar concentrations. The Ln³⁺s did not alter inhibition of I_AC by Angiotensin II or cAMP. Ln³⁺-induced uncoupling of ACTH receptor activation from I_AC inhibition was prevented by raising the external Ca²⁺ concentration from 2 to 10 mM. The divalent cation Ni²⁺ (500 µM) also blocked ACTH-dependent inhibition of I_AC through a Ca²⁺-sensitive mechanism. The results are consistent with a model in which Ln³⁺s produce opposing actions on I_AC K⁺ currents through two separate binding sites. In addition to directly inhibiting I_AC, Ln³⁺s (and Ni²⁺) bind with high affinity to a Ca²⁺-selective site associated with the ACTH receptor. By displacing Ca²⁺ from this site, Ln³⁺s prevent ACTH from binding and accelerate its dissociation. These results identify Ln³⁺s as a relatively potent group of non-competitive ACTH receptor antagonists. Allosteric actions of trivalent and divalent metal cations on hormone binding, mediated through Ca²⁺ specific sites, may be common to a variety of peptide hormone receptors.