Bovine pancreatic ribonuclease A (RNase A) is a much studied enzyme that efficiently catalyzes the cleavage of RNA. The active site of RNase A contains two histidine residues with imidazole groups positioned to act as a general base (H12) and a general acid (H119) during catalysis of RNA cleavage. Recombinant DNA techniques were used to produce mutant enzymes in which either H12 or H119 was replaced with an alanine residue. Each mutation resulted in a 104-fold decrease in the value of kcat/Km for cleaving either poly(C) or UpA. Thus, H12 and H119 each lower by 5–6 kcal/mol the free energy of the rate-limiting transition state during RNA cleavage. The value of kcat/Km for cleavage of UpOC6H4-p-NO2 was decreased by 104-fold by replacing H12 but was unaffected by replacing H119. This result provides the first direct evidence that H119 acts as a general acid during catalysis by RNase A.