A study of creatine kinase modification by ATP gamma-(N-(2-chloroethyl)-N-methyl)amide was performed. The attachment 1,7-1,8 moles of analogue per mole of functional dimer results in full inactivation of the enzyme. The substrates, ATP and ADP, protect the enzyme both against inactivation and covalent binding of analogue. The affinity modification rate depends on the reagent and magnesium ion concentrations and pH of the reaction mixture. The dissociation constants (1,0 and 1,5 mM) for the enzyme-analogue complexes and the affinity modification maximal rate constants (2,1 X 10(-3) and 1,2 X 10(-3) c-1) in the absence and presence of Mg2+ ions were estimated. Some differences in the affinity modification rates were observed for the nonidentical M and M'-subunits of creatine kinase. The data obtained are indicative of a histidine residue alkylation by the ATP analogue. This histidine (pK 7,7) may function as a general acid-base catalyst in deprotonation of the guanidinium group of creatine as the latter is phosphorylated by ATP.