Direct evidence is presented for a conformational switch in 16S ribosomal RNA (rRNA) that affects tRNA binding to the ribosome and decoding of messenger RNA (mRNA). These data support the hypothesis that dynamic changes in rRNA structure occur during translation. The switch involves two alternating base-paired arrangements apparently facilitated by ribosomal proteins S5 and S12, and produces significant changes in the rRNA structure. Chemical probing shows reciprocal enhancements or protections at sites in 16S rRNA that are at or very near sites that were previously crosslinked to mRNA. These data indicate that the switch affects codon-anticodon arrangement and proper selection of tRNA at the ribosomal A site, and that the switch is a fundamental mechanism in all ribosomes.