Treatment of rabbit muscle creatine kinase (EC 2.4.3.2) with either butanedione in borate buffer or phenylglyoxal in Veronal buffer decreases enzymatic activity correlating with the modification of a single arginyl residue per subunit of the dimeric enzyme. Very little activity is lost when modification is performed in the presence of MgATP or MgADP. Nucleotide binding to the modified enzyme is virtually abolished as determined by ultraviolet difference spectroscopy. The data suggest that an arginyl residue plays an essential role in the enzymatic mechanism of creatine kinase, probably as a recognition site for the negatively charged oligophosphate moiety of the nucleotide.