TRPV6, a Ca2+ specific channel, is regulated by calmodulin (CaM). We investigated in vivo the interaction of CaM and TRPV6 in response to an increase in [Ca2+]i employing patch-clamp, Fura-2 and FRET microscopy. Overexpression of CaM or its Ca2+-insensitive mutant (CaMMUT) in HEK293 cells was probed on various TRPV6-fragments to pinpoint the functionally relevant TRPV6-CaM interaction site. A significant decrease of TRPV6 Ca2+-current density was only observed with CaM in accordance with live cell FRET confocal microscopy revealing a high FRET signal that suggests a Ca2+-dependent interaction of CaM. The in vivo functionally relevant CaM interaction site was localized at the very end of the TRPV6 C-terminus (aa 695-727) as the inactivation of a TRPV6 mutant lacking these amino acids was reduced similar to TRPV6 treated with the CaM antagonist calmidazolium. Consistently, no substantial augmentation of FRET upon elevation of [Ca2+]i occurred for TRPV6D695-727 coexpressed with CaM. In contrast to Ca2+-dependent inactivation of voltage-gated L-type channels, CaM is not constitutively tethered to TRPV6, but interacts dynamically when [Ca2+]i increases thereby promoting Ca2+ current inactivation. Supported by FWF P-15387, P-16537