It has been previously reported that, with a fluorescence probe formed from o-phthaldehyde (OPTA) and the thiol and amino groups at or near the active site of creatine kinase, inactivation and exposure of the probe take place simultaneously and well before unfolding of the molecule as a whole. In this study, the inactivation and modification kinetics of purified rabbit muscle creatine kinase by OPTA have been compared. the former by following the substrate reaction in the presence of a previously described inactivator. The microscopic rate constants for the reaction of the inactivator with the free enzyme and with the enzyme-substrate complexes were determined. From the results obtained it appears that OPTA is noncompetitive with respect to both substrates. The inactivation kinetics is monophasic with OPTA, and neither ATP nor creatine alone affect the rate constant of inactivation of the enzyme, indicating that the irreversible inhibition of creatine kinase by OPTA is of the noncompetitive type.