In an attempt to produce a bovine pancreatic ribonuclease A (RNase A) with increased activity and stability, the catalytic pair of His12 and His119 was substituted with aspartic acid or glutamic acid, and aspartic acid, respectively, to evaluate the role of the two histidine residues in the activity and stability. Kinetic analysis revealed that k(cat)/K-m values were significantly reduced for all mutant enzymes due to a decreased k(cat) rather than an increased K-m: the k(cat) values for both CpA and C>p of H12D and H12E decreased to about 1/1000; the k(cat) values of H119D decreased by 1/3300 for CpA and 1/80 for C>p. Thus, neither Asp nor Glu is able to act solely as an efficient catalytic residue of RNase A. Alkylation with iodoacetic acid (IAA) revealed that mutant enzymes had reduced reaction rates and that no modification was evident at Glu12 and Asp12 of H12E and H12D, respectively. This indicates that the low catalytic activity of mutant enzymes could be due to low basicity of Asp12 and Glu12. While the T-m of H119D was almost the same as that of the wild-type enzyme, the T-m of both H12D and H12E markedly decreased. It became apparent that His12 located at the bottom of the active site cleft contributes significantly to the structural stability of RNase A.