Following 30 weeks of exposure to 0.3% ethionine in the diet, male Fischer rats bearing liver cell tumors showed a marked increase (9.5 fold) in hepatic metallothionein (MT) concentrations relative to control, as assessed by the cadmium-hemoglobin assay. To confirm the presence of MT in the tumor-containing livers of ethionine-fed rats, biochemical analyses were performed. Spectral analysis of a purified protein from ethionine-treated livers revealed a UV absorption spectrum characteristic of MT. Gel filtration (Sephadex G-75) indicated a prominent copper (Cu) and zinc (Zn)-containing peak with a Mr (approx. 9 000) similar to that of MT. Concurrent with the elevation in hepatic MT in ethionine-fed rats there was an increase in the concentration of Cu and Zn in hepatic cytosol.