The polar microenvironment around the reactive Cys283 of rabbit muscle creatine kinase was explored using kinetic analysis of substrates reaction in the presence of modifiers. In the present study, three specific sulphydryl reagents, 5,5'-dithiobis(2-nitrobenzoic acid), 6,6'-dithiodinicotinic acid and 2,2'-dithiodipyridine, were applied as modifiers to react with Cys283 of creatine kinase. The inactivation kinetics of creatine kinase by the modifiers was analyzed. The microscopic rate constants for reactions of the modifiers with free enzyme and enzyme-substrate complexes were also determined. The results suggested that the inactivation rate of creatine kinase by 5,5'-dithiobis(2-nitrobenzoic acid) was the fastest, followed by 6,6'-dithiodinicotinic acid and then 2,2'-dithiodipyridine. Interestingly, 5,5'-dithiobis(2-nitrobenzoic acid) and 6,6'-dithiodinicotinic acid functioned as non-complexing modifiers, while 2,2'-dithiodipyridine did a complexing modifier. The results here indicated that the electrophilic group was predominant around Cys283, and that the presence of substrates seemed to have different effects on the inactivation reactions of creatine kinase by the three modifiers. Furthermore, the findings in this study may provide a novel explanation for the low pKa value of Cys283.