Abstract  The recently identified TMEM16/anoctamin protein family includes Ca²⁺‐activated anion channels (TMEM16A, TMEM16B), a cation channel (TMEM16F) and proteins with unclear function. TMEM16 channels consist of eight putative transmembrane domains (TMs) with TM5–TM6 flanking a re‐entrant loop thought to form the pore. In TMEM16A this region has also been suggested to contain residues involved in Ca²⁺ binding. The role of the putative pore‐loop of TMEM16 channels was investigated using a chimeric approach. Heterologous expression of either TMEM16A or TMEM16B resulted in whole‐cell anion currents with very similar conduction properties but distinct kinetics and degrees of sensitivity to Ca²⁺. Furthermore, whole‐cell currents mediated by TMEM16A channels were ∼six times larger than TMEM16B‐mediated currents. Replacement of the putative pore‐loop of TMEM16A with that of TMEM16B (TMEM16A‐B channels) reduced the currents by ∼six‐fold, while the opposite modification (TMEM16B‐A channels) produced a ∼six‐fold increase in the currents. Unexpectedly, these changes were not secondary to variations in channel gating by Ca²⁺ or voltage, nor were they due to changes in single‐channel conductance. Instead, they depended on the number of functional channels present on the plasma membrane. Generation of additional, smaller chimeras within the putative pore‐loop of TMEM16A and TMEM16B led to the identification of a region containing a non‐canonical trafficking motif. Chimeras composed of the putative pore‐loop of TMEM16F transplanted into the TMEM16A protein scaffold did not conduct anions or cations. These data suggest that the putative pore‐loop does not form a complete, transferable pore domain. Furthermore, our data reveal an unexpected role for the putative pore‐loop of TMEM16A and TMEM16B channels in the control of the whole‐cell Ca²⁺‐activated Cl⁻ conductance.