The Ca2+-activated chloride channel, TMEM16A, plays an important role in smooth muscle contraction, sensory neuron excitability and fluid secretion. Blockers of the channel like niflumic acid (NFA), A9C and CaCCinh-A01 have been characterized previously [1] but the exact binding mechanism of these drugs has not been studied. In the present study, mutations of different regions of the TMEM16A channel were investigated and the effect of niflumic acid and CaCCinh-A01 observed. Resultant currents were studied using the whole-cell patch clamp technique from HEK293 cells expressing the wild-type (WT) or mutant channels. For comparison, currents evoked at +100 mV are reported as mean values ± S.E.M. TMEM16A assembles as a dimer with each monomer unit containing an individual pore and ten transmembrane alpha helices [2]. Point mutation of the Ca2+-binding residue E702A on the alpha 7 transmembrane resulted in Ca2+-insensitive channels. Application of 30 μM NFA to the WT currents reduced the current amplitude from 2.06 ± 0.24 nA to 0.73 ± 0.13 nA (n=6, p<0.05). However, in the E702A mutant, the effect of NFA was reversed, with it acting as an activator, increasing the current amplitude from 0.58 ± 0.08 to 1.36 ± 0.32 nA (n=6, p<0.05). The effect of CaCCinh-A01 (10 μM) did not change, with mutant E702A currents being blocked from 1.06 ± 0.19 to 0.29 ±0.06 nA (n=6, p<0.05). CaCCinh-A01 (10 μM) reduced current amplitude in the WT from 3.15 ± 0.58 to 0.74 ± 0.17 nA (n=6, p<0.05). Three pore mutations R535A, R515A and K588A [3] situated in α3-α4 linker, α3 and α5 respectively were constructed and the effect of 10 μM CaCCinh-A01 compared. The blocking effect of CaCCinh-A01 was markedly reduced in the R535A mutant (from 3.08 ± 0.47 to 1.62 ± 0.22 nA, n=6, p>0.05), suggesting it might possibly be a binding site residue for the drug. However, 30 μM NFA blocked R535A mutant currents similar to WT (from 1.69 ± 0.18 to 0.66 ± 0.09 nA, n=6,p<0.05). The R515A mutant resulted in channels with altered kinetics and neither NFA, nor CaCCinh-A01, were able to effectively block these currents (e.g. 10 μM CaCCinh-A01 reduced them only from 0.85 ± 0.14 to 0.72 ± 0.13 nA, n=6, p>0.05; 30 μM NFA reduced them from 1.08 ± 0.24 to 0.79 ± 0.18 nA, n=6, p>0.05). The effect of CaCCinh-A01 was not altered on the K588A mutant, with the channels being blocked as in the WT. These results reflect two distinct regions in the TMEM16A channel being possible binding pockets for NFA and CaCCinh-A01. The Ca2+-binding region may be contributing to the functionality of NFA, as mutations in that region reversed its action, while the pore might be the binding region for CaCCinh-A01.