Fluoride is an ubiquitous environmental ion. Under mildly acidic conditions, the weakly acidic character of hydrofluoric acid allows it to pass through the cell membrane and accumulate F- to toxic levels. One mechanism developed to mitigate this problem is the Fluc family of fluoride channels. These channels, extremely selective for fluoride over chloride and other anions, allow passive draining of fluoride down to sub-toxic levels. Recent structural work on two homologues of these proteins has demonstrated some very unusual characteristics. Like the small multidrug resistance transporter EmrE, Fluc is a dual-topology membrane protein, where the functional channel is an antiparallel homodimer made up of two Fluc molecules inserted into the membrane in opposite orientations. Surprisingly, structures of the Fluc proteins do not show a single, clear pore through the center of the dimer. Rather, there appears to be two independent ion pathways through the channel, with residues from both protein chains contributing to both pathways. We are currently using a combination of mutagenesis, electrophysiology, and crystallography to attempt to clearly delineate the pathways of ion conduction through the channel, and try to determine conclusively whether Fluc has a “double-barreled”, parallel channel architecture. To that end, we have produced a functional Fluc concatomer, with both members of the antiparallel dimer contained in a single protein chain, linked by an additional transmembrane helix to maintain the antiparallel structure. This construct has allowed us to mutagenically degrade each pore independently, and thus demonstrate the presence of two independent pores through this small channel.