The principle of this assay can be interpreted by comparing the rates of HCl efflux (or Cl -/OH -antiport) and HX influx (or Cl -/X -antiport). In the case of HX influx being more efficient than HCl efflux, the interior of the vesicles is acidified, inferring X -> Cl -selectivity and membrane permeability of the transporter•anion complex. In contrast, basification of the internal solution due to faster HCl efflux by anions other than Cl -indicates a higher transporter-facilitated membrane permeability of Cl -over X -. The selectivity between two non-chloride anions X -and Y -can also be determined by comparing the maximum ΔpHIN (maximum intravesicular pH change in the kinetic plots) values under NaX IN /NaCl OUT and NaY IN /NaCl OUT conditions. If the transporter does not facilitate H + /OH -transport, leading to no activity in the HPTS assay, a proton channel such as FCCP (or CCCP) can be added.