It has been shown that the active dicyano derivative of creatine kinase (ATP:creatine N-phosphotransferase) obtained by cyanolysis of the 5,5'-dithiobis(2-nitrobenzoic acid)-modified and inactivated enzyme contains, as does the native enzyme, two reactive SH groups. Modification of these two SH groups leads to complete inactivation of the dicyano enzyme. Reaction with 4-iodoacetamido-1-naphthol introduces fluorescent labels at these reactive SH groups of the native and the dicyano enzymes. Following tryptic digestion, the respective fluorescent-labelled peptides have been separated by HPLC and the amino acid composition analysis of these peptides has shown that they are consistent with the sequence of the peptide segment containing the active-site SH of Cys-282 of creatine kinase for both the native and the dicyano enzymes, showing that the active SH groups are free in the dicyano enzyme. Upon mild denaturation in 3 M urea, it can be shown that two of the SH groups partially buried in the native enzyme have been cyanylated in the dicyano enzyme. The two reactive SH groups are therefore essential for the activity of creatine kinase and the two cyanylated SH groups are internal groups which probably contributes partially to the stabilization of an active conformation of the enzyme molecule.