Phospholipid scrambling, the process by which cells collapse the lipid asymmetry on the plasma membrane, causes phosphatidylserine (PS) to be exposed to the outer leaflet of the plasma membrane, triggering blood coagulation and marking apoptotic cells. TMEM16 proteins were originally thought to be ion channels, but it quickly became clear that this family of membrane proteins was characterized by a high degree of functional divergence when other members were suggested to be Ca2+-dependent phospholipid scramblases. These findings created a controversy in the field, especially around the function of TMEM16F which was reported to be an ion channel, a phospholipid scramblase and both. To solve this controversy we decided to take a biochemical approach, aiming to purify TMEM16 members for functional reconstitution into artificial membranes. We identified a novel TMEM16 homologue, afTMEM16 from the fungus Aspergillus fumigatus, which is a dual function, Ca2+-dependent channel/scramblase where the two functions are regulated by the same Ca2+-binding site. This was the first direct evidence that TMEM16 members are indeed phospholipid scramblases and that TMEM16s can be dual function proteins. Shortly after, the crystal structure of a fungal TMEM16 phospholipid scramblase, nhTMEM16, was solved by the Dutzler group. The structure provided the basis for a first mechanistic hypothesis on lipid translocation: interactions between the headgroup of a phospholipid and residues within a 8-11 Å wide cavity at the protein-lipid interface would allow lipids to get translocated across the membrane in a semicircular, bi-directional fashion. We decided to begin testing this hypothesis by investigating how lipids of increasing headgroup size are scrambled in the presence and absence of Ca2+. We found that in the Ca2+-bound state the headgroup size cut off is considerably larger than the width of the cavity, suggesting that tight interactions between the lipid headgroup and the cavity are not required for scrambling. Interestingly, we found that the apo-state conductance has a similar cut off than that of the Ca2+-bound state, suggesting the presence of one conductive state where the apo state can visit the open conformation even in the absence of Ca2+.