The catalytic activity of adenosine kinase (EC 2.7.1.20) from yeast is very labile. Even incubation with thiols provokes a loss of two thirds of its enzymatic activity. Concomitantly, two SH-groups appear on the enzyme in addition to the single SH-group already present in the untreated enzyme, the latter being absolutely essential for activity. Treatment of adenosine kinase with thiols does not substantially affect the binding of the substrates adenosine and ATP-Mg2. The reactivity of the two newly formed SH-groups is diminished in the presence of ATP-Mg2, whereas adenosine has no influence. The opposite holds for the reactivity of the single SH-group essential for enzymatic activity. Complete reactivation of the enzymatic activity after incubation of adenosine kinase with thiols can be achieved by reoxidation of the enzyme in presence of high concentrations of adenosine. These observations suggest the notion that adenosine kinase contains an essential SH-group close to the adenosine-binding site and a disulfide bridge near to the binding site of ATP-Mg2, the latter being easily accesible to the reduction by thiols.