The properties of SH-groups of mitochondrial creatine kinase existing in solution as a hexamer with Mr of (240 +/- 12) X 10(3) Da, were investigated. The number and reactivity of SH-groups by specific modifiers--[5.5'-dithiobis-(2-nitrobenzoic acid), DTNB; 7-chloro-4-nitrobenzo-2-oxo-1.3-diazol, NBD-Cl; 2.2'-dithiopyridine, DTP] were determined. It was found that each subunit of the enzyme hexameric molecule contains two modified SH-groups, only one of which is protected against modification by Mg-ADP, Mg-ATP as well as during the formation of the transition state analog (TSA)--E-Mg X ADP-NO3-creatine--and is essential for the enzyme activity. These six essential SH-groups within the hexameric molecule of mitochondrial creatine kinase may be classified into two groups according to the rate of their interaction with DTNB, NBD-Cl and DTP. The rate constants of modification of three fast and three slow essential SH-groups differ 4-10 times. The kinetics of enzyme inactivation by iodoacetamide (IAA) is biphasic; each phase is characterized by a 50% loss of activity. The inactivation constants differ 30 times; both phases being protected by TSA; consequently, the inactivation is caused by the binding of IAA to the essential SH-groups. The unequal reactivity of essential SH-groups seems to be preexisting. Using a computer analysis, the dependence of the amount of residual activity on the number of modified SH-groups by NBD-Cl and DTNB was studied. The interaction of NBD-Cl and DTNB with the most reactive essential SH-groups in half of the subunits results in the inactivation of these subunits as well as in partial or complete inactivation of the other half of the non-modified subunits. The degree of inactivation of the latter 50% of subunits strongly depends on the nature of the modifier. The inactivating effect of the bound modifier is translated from one subunit to another in one direction. The experimental results point to asymmetrical association of mitochondrial creatine kinase subunits.