The 3′ splice site of group I introns is defined, in part, by base pairs between the intron core and residues just upstream of the splice site, referred to as P9.0. We have studied the specificity imparted by P9.0 using the well-characterized L–21 ScaI ribozyme from Tetrahymena by adding residues to the 5′ end of the guanosine (G) that functions as a nucleophile in the oligonucleotide cleavage reaction: CCCUCUA5 (S) + NNG [lhard ][rharu ] CCCUCU + NNGA5. UCG, predicted to form two base pairs in P9.0, reacts with a (kcat/KM) value ∼10-fold greater than G, consistent with previous results. Altering the bases that form P9.0 in both the trinucleotide G analog and the ribozyme affects the specificity in the manner predicted for base-pairing. Strikingly, oligonucleotides incapable of forming P9.0 react ∼10-fold more slowly than G, for which the mispaired residues are simply absent. The observed specificity is consistent with a model in which the P9.0 site is sterically restricted such that an energetic penalty, not present for G, must be overcome by G analogs with 5′ extensions. Shortening S to include only one residue 3′ of the cleavage site (CCCUCUA) eliminates this penalty and uniformly enhances the reactions of matched and mismatched oligonucleotides relative to guanosine. These results suggest that the 3′ portion of S occupies the P9.0 site, sterically interfering with binding of G analogs with 5′ extensions. Similar steric effects may more generally allow structured RNAs to avoid formation of incorrect contacts, thereby helping to avoid kinetic traps during folding and enhancing cooperative formation of the correct structure.