Bovine pancreatic ribonuclease A (RNase A) catalyzes the cleavage of P–O5′ bonds in RNA. Structural analyses had suggested that the active-site lysine residue (K41) may interact preferentially with the transition state for covalent bond cleavage, thus facilitating catalysis. Here, site-directed mutagenesis and semisynthesis were combined to probe the role of K41 in the catalysis of RNA cleavage. Recombinant DNA techniques were used to replace K41 with an arginine residue (K41R) and with a cysteine residue (K41C), which had the only sulfhydryl group in the native protein. The value of kcat/Km for cleavage of poly(C) by K41C RNase was 105-fold lower than that by the wild-type enzyme. The sulfhydryl group of K41C RNase A was alkylated with 5 different haloalkylamines. The value of kcat/Km for the resulting semisynthetic enzymes and K41R RNase A were correlated inversely with the values of pKa for the side chain of residue 41. Further, no significant catalytic advantage was gained by side chains that could donate a second hydrogen bond. These results indicate that residue 41 donates a single hydrogen bond to the rate-limiting transition state during catalysis.