MT (metallothionein), metal-binding protein, was induced with Cd and isolated from rat liver supernatant by heat-treatment, Sephadex G-50 gel filtration, and DEAE Sephadex A-25 ion exchange chromatography. Two isoproteins, MT-A and MT-B, were separated with overall yield of 30%. In order to characterize the chemical properties of metal-binding sites of MT, the cysteinyl groups were examined with DTNB. The reaction of MT-A with DTNB was dependent on the pH and ionic strength. The plot of pseudofirst order rate constant against pH showed a minimum near pH 7.5. It was also found that the DTNB-titrable thiol groups of MT-A were markedly affected by either the presence of metal or by the changes of protein conformation, thus this protein appears to contain at least two different types of cadmium-sulfur bonding.