RNA cleavage by the Tetrahymena ribozyme requires recognition of the reaction-site helix by the catalytic apparatus. This binding can occur in several registers, each of which results in reaction at a different nucleotide in the helix. We now identify commensurate sets of 2'-hydroxyl interactions on both strands of the reaction-site helix that account for its translocation into alternative binding registers. These results indicate that the ribozyme has a relatively rigid substrate-binding pocket into which the helix can bind in different alignments. A similar mechanism of reaction site recognition is proposed to occur during intron circularization and ribozyme polymerase activity. Translocation of the reaction site duplex provides an example of structural heterogeneity in packing of helices during the tertiary folding of RNA.