Fundamental nature of the metallothionein fraction, effect of some compounds, and binding ability with various metals were examined in vitro, using the supernatant of liver homogenate of rabbits dosed with cadmium, and the following results were obtained. 1) Metallothionein fraction showed a strong resistance to heat and treatment with organic solvents. 2) o-Iodosobenzoic acid, sodium diethyl dithiocarbamate and ethylenediaminetetraacetic acid (EDTA) completely liberated cadmium and zinc in the metallothionein fraction, and D-penicillamine lowered zinc content to 88%. 3) Copper and silver underwent substitution with cadmium and zinc in the metallothionein fraction, and cadmium and mercury substituted with zinc. Lead liberated only zinc. Fifteen other metals examined did not show any effect. 4) Binding ability of metallothionein with metals decreased in the order of copper, cadmium, mercury, and zinc under the present experimental conditions. 5) Substitution of zinc in the metallothionein fraction with cadmium occurred even in as low a concentration of cadmium chloride solution as 8.9×10^-6M. There was no difference in the DEAE-cellulose column chromatographic behavior between the metallothionein fraction in which zinc was substituted with cadmium and that without such substitution.