S-Adenosylhomocysteine (AdoHcy) hydrolase regulates biomethylation and homocysteine metabolism. It has been proposed to be a copper binding protein playing an important role in copper transport and distribution. In the present work, the kinetics of binding and releasing of copper ions was studied using fluorescence method. The dissociation constant for copper ions with AdoHcy hydrolase was determined by fluorescence quenching titration and activity titration methods using ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), and glycine as competitive chelators. The experimental results showed that copper ions bind to AdoHcy hydrolase with a Kd of～10-11M. The association rate constant was determined to be 7×10-6 M-1 s-1. The releasing of copper ions from the enzyme was found to be biphasic with a k(1) of 2.8×10-3 s-1 and k(2) of 1.7×10-5 s-1. It is suggested that copper ions do not bind to the substrate binding sites because the addition of adenine substrate did not compete with the binding of copper to AdoHcy hydrolase. Interestingly, it was observed that EDTA could bind to AdoHcy hydrolase with a dissociation constant of K1=8.0×10-5M and result in an increased affinity (Kd=～10-17M) of binding of copper ions to the enzyme.