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Riboswitches are gene-regulatory RNA motifs located in the 5' untranslated regions of certain bacterial mRNA's. Riboswitches regulate gene expression by binding a metabolite related to the downstream gene, causing a conformational change that alters the accessibility of the gene for either transcription or translation. An important class of riboswitches that bind fluoride (F-) has been identified recently in bacteria and archaea, shedding light on how these organisms regulate internal fluoride concentrations to mitigate toxicity. The crystal structure of the fluoride riboswitch from Thermophilus petrophila shows a binding pocket in which the F- ion is coordinated by three Mg2+ ions. However, how ligand recognition and RNA folding are coupled to selectively encapsulate F- is not fully understood. Here, single-molecule TIRF microscopy and FRET are used to gain insights into the functional dynamics of fluoride riboswitches. Individual fluorescently-labelled fluoride riboswitches are immobilized on a quartz microscope slide, and the change in FRET efficiency between the fluorophors is used to study the ligand-binding mechanism and other cation- or denaturant-dependent structural transitions.