TMEM16A and TMEM16B proteins are Ca2+ activated Cl- channels (CaCCs) with eight putative transmembrane segments. As shown previously, expression of TMEM16B generates CaCCs characterized by a ten-fold lower Ca2+ affinity and by faster activation and deactivation kinetics with respect to TMEM16A. To investigate the basis of the different properties, we generated chimeric proteins in which different domains of the TMEM16A protein were replaced by equivalent domains of TMEM16B. Replacement of the N-terminus, transmenbrane domains 1 and 2 (TMD1-2), the first intracellular loop, and TMD3-4 did not change channel properties. Instead, replacement of the intracellular loop 3, decreased the apparent Ca2+ affinity by nearly eight-fold with respect to wild type TMEM16A. In contrast, the membrane currents derived from chimeras having the TMD7-8 or the carboxy-terminus of TMEM16B showed faster activation and deactivation rates without a change in Ca2+-sensitivity. Significantly accelerated kinetics were also found when the entire carboxy-terminus of the TMEM16A protein (77 amino acid residues) was deleted. Our findings indicate that the third intracellular loop of TMEM16A and TMEM16B protein is the site involved in Ca2+-sensitivity, whereas the carboxy-terminal part, including transmembrane domains 7 and 8, affect the rate of transition between the open and the closed state.