Riboswitches are genetic control elements located mainly within the 5' untranslated regions of messenger RNAs. These RNA elements undergo conformational changes that modulate gene expression upon binding of regulatory signals including vitamins, amino acids, nucleobases and uncharged tRNA. The thiamin pyrophosphate (TPP)-binding riboswitch (THI-box) is found in all three kingdoms of life and can regulate gene expression at the levels of premature termination of transcription, initiation of translation and mRNA splicing. The THI-box is composed of two parallel stacked helices bound by another helix in a three-way junction. We performed an in vivo expression analysis of mutants with substitutions in conserved bases located at the interior and terminal loops of the Escherichia coli thiM THI-box, which is translationally regulated, and observed two different phenotypic classes. One class exhibited high expression during growth in the presence or absence of thiamin, while the second class exhibited low expression regardless of the presence of thiamin. Accessibility of the Shine-Dalgarno region of the RNA following the addition of TPP was monitored by means of an oligonucleotide-dependent RNase H cleavage assay, and binding of 30S ribosomal subunits. These studies showed that high- and low-expression mutant RNAs are locked in the non-repressive and repressive conformations respectively.