It has been established that Tyr-42, Tyr-45, and Glu-46 take part in a structural motif that renders guanine specificity to ribonuclease T1. We report on the impact of Tyr-42, Tyr-45, and Glu-46 substitutions on the guanine specificity of RNase T1. The Y42A and E46A mutations profoundly affect substrate binding. No such effect is observed for Y45A RNase T1. From the kinetics of the Y42A/Y45A and Y42A/E46A double mutants, we conclude that these pairs of residues contribute to guanine specificity in a mutually independent way. From our results, it appears that the energetic contribution of aromatic face-to-face stacking interactions may be significant if polycyclic molecules, such as guanine, are involved.