The anoctamins are a newly described family of anion channels. It has been proposed that anoctamin-1 (Ano1) has 8 transmembrane segments (TMDs) with a re-entrant loop between the 5th and 6th TMD that participates in forming the selectivity filter. To clarify the detailed structure and function of this region, we used cysteine accessibility scanning mutagenesis. Among 16 endogenous cysteines in mAno1, C370, C379, C383, C386, C395, and C836, which are predicted to be in the first and last extracellular loops, are essential for mAno1 function. The construct containing the 6 essential cysteines (mAno16C) was not significantly affected by extracellularly applied, membrane-impermeant MTSET or MTSES. mAno16C was used as a template cysteine scanning mutagenesis of the re-entrant loop (620 - 668). The accessibility of the cysteine-substituted amino acids to MTS reagents were determined as well as the effects on the relative permeability and conductance of the channel to anions. Cysteines introduced at positions 620-626 reduced current amplitude significantly and neither MTSET nor MTSET had significant effects. Cysteine substitution of amino acids 628 - 632 produced currents that were rapidly affected by extracellular MTSET and/or MTSES, suggesting that amino acids 628-632 are near the outer mouth of the channel. Cysteine substitution of amino acids 634 −662 were not sensitive to MTS reagents although some of these substitutions produced non-functional channels. The ionic selectivity of various mutations in this region were also examined. Further, the accessibility of HA epitopes introduced at various positions near the re-entrant loop was examined to help establish the topology of the channel. These data support the suggestion that amino acids 628-632 may contribute to the outer mouth of the pore, but it remains uncertain whether the re-entrant loop forms the selectivity filter of the channel.