The Ca(2+)-selective TRPV6 as well as the L-type Ca(2+) channel are regulated by the Ca(2+)-binding protein calmodulin (CaM). Here, we investigated the interaction of CaM with rat (r)TRPV6 in response to alterations of intracellular Ca(2+), employing Ca(2+)-imaging and patch-clamp techniques. Additionally, confocal Förster resonance energy transfer (FRET) microscopy on living cells was utilized as a key method to visualize in vivo protein-protein interactions essential for CaM regulation of rTRPV6 activity. The effects of overexpressed CaM or its Ca(2+)-insensitive mutant (CaM(MUT)) was probed on various rTRPV6 mutants and fragments in an attempt to elucidate the molecular mechanism of Ca(2+)/CaM-dependent regulation and to pinpoint the physiologically relevant rTRPV6-CaM interaction site. A significant reduction of rTRPV6 activity, as well as an increase in current inactivation, were observed when CaM was overexpressed in addition to endogenous CaM. The Ca(2+)-insensitive CaM(MUT), however, failed to affect rTRPV6-derived currents. Accordingly, live cell confocal FRET microscopy revealed a robust interaction for CaM but not CaM(MUT) with rTRPV6, suggesting a strict Ca(2+) dependence for their association. Indeed, interaction of rTRPV6 or its C terminus with CaM increased with rising intracellular Ca(2+) levels, as observed by dynamic FRET measurements. An rTRPV6Delta(695-727) mutant with the very C-terminal end deleted, yielded Ca(2+) currents with a markedly reduced inactivation in accordance with a lack of CaM interaction as substantiated by FRET microscopy. These results, in contrast with those for CaM-dependent L-type Ca(2+) channel inactivation, demonstrate a dynamic association of CaM with the very C-terminal end of rTRPV6 (aa 695-727), and this enables acceleration of the rate of rTRPV6 current inactivation with increasing intracellular CaM concentrations.