The SLC26 gene family encodes proteins with diverse functions in numerous tissues and organs. Some mammalian SLC26 isoforms function as anion exchangers, while others are anion channels (SLC26A7) or membrane-bound motor proteins (SLC26A5/prestin). At present, little is known about the molecular basis of this functional variability. We here studied the subunit stoichiometry of bacterial, teleost and mammalian SLC26 isoforms using blue native polyacrylamide gel electrophoresis, expression of concatanated transporters, chemical cross-linking and measurements of voltage-dependent capacitances after expression in Xenopus oocytes or in mammalian cells. All tested SLC26s are assembled as dimers composed of two identical subunits. Overexpression and purification of a prokaryotic isoform from P. aeruginosa in E. coli yielded in a homogenous population of dimeric proteins. Co-expression of two mutant SLC26A5/prestins resulted in motor proteins with novel electrical properties, indicating that the two subunits do not function independently. Our results demonstrate that an evolutionarily conserved dimeric quaternary structure represents the native and functional state of SLC26 transporters.