RNase T is a classical member of the DEDDh family of exonucleases with a unique sequence preference in that its 3′-to-5′ exonuclease activity is blocked by a 3′-terminal dinucleotide CC in digesting both single-stranded RNA and DNA. Our previous crystal structure analysis of RNase T-DNA complexes show that four phenylalanine residues, F29, F77, F124, and F146, stack with the two 3′-terminal nucleobases. To elucidate if the π–π stacking interactions between aromatic residues and nucleobases play a critical role in sequence-specific protein–nucleic acid recognition, here we mutated two to four of the phenylalanine residues in RNase T to tryptophan (W mutants) and tyrosine (Y mutants). TheEscherichia colistrains expressing either the W mutants or the Y mutants had slow growth phenotypes, suggesting that all of these mutants could not fully substitute the function of the wild-type RNase Tin vivo. DNA digestion assays revealed W mutants shared similar sequence specificity with wild-type RNase T. However, the Y mutants exhibited altered sequence-dependent activity, digesting ssDNA with both 3′-end CC and GG sequences. Moreover, the W and Y mutants had reduced DNA-binding activity and lower thermal stability as compared to wild-type RNase T. Taken together, our results suggest that the four phenylalanine residues in RNase T not only play critical roles in sequence-specific recognition, but also in overall protein stability. Our results provide the first evidence showing that the π−π stacking interactions between nucleobases and protein aromatic residues may guide the sequence-specific activity for DNA and RNA enzymes.