Ligand binding sites within proteins can interact by allosteric mechanisms to modulate binding affinities and control protein function. Here we present crystal structures of the regulator of K(+) conductance (RCK) domain from a K(+) channel, MthK, which reveal the structural basis of allosteric coupling between two Ca(2+) regulatory sites within the domain. Comparison of RCK domain crystal structures in a range of conformations and with different numbers of regulatory Ca(2+) ions bound, combined with complementary electrophysiological analysis of channel gating, suggests chemical interactions that are important for modulation of ligand binding and subsequent channel opening.