TMEM16F, also known as Anoctamin 6 (ANO6), is a membrane protein that has been associated with phospholipid scramblase and ion channel activity. However, the characteristics of TMEM16F-dependent channels, particularly the ion selectivity, are a matter of debate. Furthermore, the direct involvement of TMEM16F in phospholipid scrambling has been questioned. We studied the properties of different TMEM16F isoforms generated by alternative splicing. Using whole-cell patch-clamp recordings, we found that V1, V2 and V5 variants generated membrane currents activated by very high (micromolar) intracellular calcium concentrations and positive membrane potentials. These variants showed different degrees of calcium sensitivity and kinetics of activation but similar ion permeability, characterized by a slight selectivity for chloride over sodium. A fourth variant (V3) showing a unique carboxy-terminus was devoid of activity, in agreement with its intracellular localization. We also measured scramblase activity using the binding of annexin V to detect phosphatidylserine on the cell surface. V1, V2 and V5 variants were associated with calcium-dependent phosphatidylserine externalization. However, a significant difference was noted between ion transport and lipid scrambling. In intact cells, increasing intracellular calcium with 1 µM ionomycin markedly stimulated lipid scrambling but not anion transport. A modest level of anion transport was observed only with 10 µM ionomycin. Interestingly, introduction of an activating mutation, D409G, produced a marked increase in the apparent calcium sensitivity of TMEM16F-dependent channels. In parallel, this mutation also enhanced the extent of phosphatidylserine externalization that occurred even under resting conditions. These results support the conclusion that TMEM16F proteins are directly involved in dual activity, as a phospholipid scramblase and as an ion channel. However, the latter function is detectable only under extreme non-physiological conditions.