Regulator of K ⁺ conductance (RCK) domains control the activity of a variety of K ⁺ transporters and channels, including the human large conductance Ca ²⁺ -activated K ⁺ channel that is important for blood pressure regulation and control of neuronal firing, and MthK, a prokaryotic Ca ²⁺ -gated K ⁺ channel that has yielded structural insight toward mechanisms of RCK domain-controlled channel gating. In MthK, a gating ring of eight RCK domains regulates channel activation by Ca ²⁺ . Here, using electrophysiology and X-ray crystallography, we show that each RCK domain contributes to three different regulatory Ca ²⁺ -binding sites, two of which are located at the interfaces between adjacent RCK domains. The additional Ca ²⁺ -binding sites, resulting in a stoichiometry of 24 Ca ²⁺ ions per channel, is consistent with the steep relation between [Ca ²⁺ ] and MthK channel activity. Comparison of Ca ²⁺ -bound and unliganded RCK domains suggests a physical mechanism for Ca ²⁺ -dependent conformational changes that underlie gating in this class of channels.