Objective: Chloride channels play fundamental roles in many physiological processes, including transepithelial fluid secretion, sensory transduction and smooth muscle and neuronal excitability. Recently, TMEM16A was associated with Ca²⁺ activated Cl^- currents (CaCC) (Yang et al., 2008; Caputo et al., 2008; Schroeder et al., 2008. Other members of the 10 protein TMEM16 family, which share a putative eight transmembrane domain topology, may also induce Cl^- currents. Here we show that mouse TMEM16F induces a CaCC when expressed in HEK293. Methods: Mouse TMEM16F was cloned from Ehrlich Ascites Tumor Cells (EATC) and expressed in its native form in HEK293 for whole cell patch recording or fused to GFP for localization studies. Mutations for functional studies where introduced by site-directed mutagenesis. Knock-down in EATC was achieved using microRNAi. Results: When expressed in HEK293 cells, mTMEM16F localized to the plasma membrane and induced outwardly rectifying currents when activated by intracellular Ca²⁺. TMEM16F induced currents showed classical CaCC features such as time-dependent activation at high positive potentials and an Eisenmann I anion selectivity sequence of SCN^- > I^- > Br^- > Cl^- > Asp. The EC₅₀ for Ca²⁺-activation was estimated at [asymp]0,7 mM with currents being inhibited by tamoxifen, niflumic acid, NS3728 and DIDS. Mutations of positively charged amino acids in the proposed re-entrant pore loop between TM5 and TM6 (R592E, K616E and K636E), abolished the TMEM16F induced CaCC, while mutation of conserved cysteines (C352A and C596A) reduced CaCC. Furthermore, stable knock-down of endogenous mTMEM16F in EATC using microRNA expressing constructs also reduced CaCC significantly. Conclusion: mTMEM16F appears to function as a CaCC in EATC and when expressed in HEK293 cells. Refs: Caputo A, Caci E et al. Science 2008; 322:590- 594. Schroeder BC, Cheng T et al. Cell 2008; 134:1019- 1029. Yang YD, Cho H et al. Nature 2008; 455:1210-1215.