TMEM16 proteins are a recently identified protein family comprising Ca2+-activated Cl- channels that generate outwardly rectifying ionic currents in response to intracellular Ca2+ elevations. Some TMEM16 family members, such as TMEM16F/ANO6 are also essential for Ca2+-dependent phospholipid scrambling. TMEM16-like genes are present in the genomes of most eukaryotic species, the function(s) of TMEM16 family members from evolutionary ancient eukaryotes is not completely clear. Here, we provide insight into the evolution of these TMEM16 proteins by similarity searches for ancestral sequences. All eukaryotic genomes contain TMEM16 homologs, but only vertebrates have the full repertoire of ten distinct subtypes. TMEM16 homologs studied so far belong to the opisthokont branch of the phylogenetic tree, which includes the animal and fungal kingdoms. An organism outside this group is Dictyostelium discoideum, a representative of the amoebozoa group that diverged from the metazoa before fungi. We here functionally investigated the TMEM16 family member from Dictyostelium discoideum. When recombinantly expressed in HEK293 cells, DdTMEM16 induces phospholipid scrambling. However, in several electrophysiological experiments we did not find evidence for a Ca2+-activated Cl- channel function of DdTMEM16.