Shortened forms of the group I intron from Tetrahymena catalyse sequence-specific cleavage of exogenous oligonucleotide substrates. The association between RNA enzyme (ribozyme) and substrate is mediated by pairing between an internal guide sequence on the ribozyme and a complementary sequence on the substrate. RNA substrates and cleavage products associate with a binding energy greater than that of base-pairing by approximately 4 kcal-mol-1 (at 42 degrees C), whereas DNA associates with an energy around that expected for base-pairing. It has been proposed that the difference in binding affinity is due to specific 2'-OH groups on an RNA substrate forming stabilizing tertiary interactions with the core of the ribozyme, or that the RNA.RNA helix formed upon association of an RNA substrate and the ribozyme might be more stable than an RNA.DNA helix of the same sequence. To differentiate between these two models, chimaeric oligonucleotides containing deoxynucleotide residues at successive positions along the chain were synthesized, and their equilibrium binding constants for association with the ribozyme were measured directly by a new gel electrophoresis technique. We report here that most of the extra binding energy can be accounted for by discrete RNA-ribozyme interactions, the 2'-OH group on the sugar residue three nucleotides from the cleavage site contributing the most interaction energy. Thus, in addition to the well documented binding of RNA to RNA by base-pairing, 2'-OH groups within a duplex can also mediate association between RNA molecules.