The reaction of p-nitrophenyl acetate with creatine phosphokinase has been reported to inhibit this enzyme (Watts, D. C., Robin, B. R., and Crook, E. M. ( 1962), Biochem J. 82, 412). In view of earlier demonstrations that this reagent inhibits chymotrypsin and glyceraldehyde 3-phosphate dehydrogenase by a specific acetylation of the active site, it was of interest to compare in detail the reaction of p-nitrophenyl acetate with creatine phosphokinase and with these other enzymes.