Calmodulin (CaM) binds to KCNQ2-4 channels within their carboxy termini, where it regulates channel function. The existing data have not resolved the Ca²⁺-dependence of the interaction between the channels and CaM. We performed GST-pulldown assays between purified KCNQ2-4 carboxy termini and CaM proteins to determine the Ca²⁺-dependence of the interaction in vitro. The assays showed substantial Ca²⁺ dependence to the interaction of the channels with wt CaM, but not with DN CaM. To demonstrate CaM/channel interactions in individual living cells, we performed fluorescence resonance energy transfer (FRET) between ECFP-tagged KCNQ2-4 channels and EYFP-tagged CaM expressed in CHO cells, performed under total internal reflection fluorescence (TIRF) microscopy, in which excitation light only penetrates several hundred nm into the cell, thus isolating membrane events. FRET was assayed between the channels and either wild-type (wt) or dominant-negative (DN) CaM, performed under conditions of either normal [Ca²⁺]_i, low [Ca²⁺]_i or high [Ca²⁺]_i induced by empirically-optimized bathing solutions. The FRET data suggest a strong Ca²⁺ dependence for the interaction between wt CaM and KCNQ2, but less so for KCNQ3 and KCNQ4. FRET between all KCNQ2-4 channels and DN CaM was robust, and not significantly Ca²⁺-dependent. These data show interactions between CaM and KCNQ channels in living cells, and suggest that the interactions between KCNQ2-4 channels and CaM likely have Ca²⁺-dependent and Ca²⁺-independent components.