Oligonucleotides containing 3′-S-phosphorothiolate linkages provide valuable analogues for exploring the catalytic mechanisms of enzymes and ribozymes, both to identify catalytic metal ions and to probe hydrogen-bonding interactions. Here, we have synthesized 2′-O-methyl-3′-thioguanosine to test a possible hydrogen-bonding interaction in the Tetrahymena ribozyme reaction. We developed an efficient method for the synthesis of 2′-O-methyl-3′-thioguanosine phosphoramidite in eight steps starting from 2′-O-methyl-N 2 -(isobutyryl) guanosine with 10.4% overall yield. Following incorporation into oligonucleotides using solid-phase synthesis, we used this new analogue to investigate whether the 3′-oxygen of the guanosine cofactor in the Tetrahymena ribozyme reaction serves as an acceptor for the hydrogen bond donated by the adjacent 2′-hydroxyl group. We show that regardless of whether the guanosine cofactor bears a 3′-oxygen or 3′-sulfur leaving group, replacing the adjacent 2′-hydroxyl group with a 2′-methoxy group incurs the same energetic penalty, providing evidence against an interaction. These results indicate that the hydrogen bond donated by the guanosine 2′-hydroxyl group contributes to catalytic function in a manner distinct from the U −1 2′-hydroxyl group.